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In vitro experiments have shown that the E2 protein of human papillomaviruses (HPV) binds to the upstream regulatory region (URR) of the viral genome and modulates transcription. Additionally, it seems to be a necessary component for viral DNA replication together with E1. We have developed a transgenic mouse model containing the URR region of the low-risk virus HPV11 that regulates the expression of the lacZ reporter gene. Most interestingly, in these mice, the transgene was exclusively expressed in the bulge region of the hair follicle but not in any other tissues. Further experimental data indicate that in double transgenic mice that also express the HPV11-E2 protein under the control of the Ubiquitin C-promoter, the transcription of the reporter gene is modulated. When E2 is present, the expression of the reporter gene also occurs exclusively in the bulge region of the hair follicles as it does in the single transgenic mice, but the expression of the lacZ driven by the URR is increased and the statistical spread is greater. Even if the expression of the reporter gene occurs in the hair follicles of the dorsal skin of an animal uniform, E2 obviously has the capacity for both to induce and to repress the URR activity in vivo.

Cryopreservation of genetically modified mouse lines prevents the loss of specific mutants that are of enormous scientific value for both basic and applied research. Cryopreservation of spermatozoa or preimplantation embryos enables discontinuation of breeding as well as archiving of specific lines for future studies. Regarding active inter-laboratory exchange of mutants, cryopreserved material is more advantageous to transport than live animals. However, transportation stress should not be trivialized. Security scanning of transport boxes at airports and customs, in particular, as well as additional cosmic radiation, pose a threat to undefined dosages of irradiation exposure. To simulate this, cryopreserved samples of mouse spermatozoa and preimplantation embryos were exposed to an X-ray dosage of 1 mGy in an X-ray machine. For subsequent investigation of the cell integrity of irradiated spermatozoa and embryos, spermatozoa forward motility as well as embryo developmental capacity and apoptosis values were examined and compared with nonirradiated control samples. The percentage of forward-moving spermatozoa per sample appears to be significantly reduced after irradiation exposure. The in vitro developmental capacity of preimplantation embryos as well as their relative share of apoptotic cells do not seem to be influenced by irradiation exposure. This leads to the assumption that, at least in preimplantation embryos, X-ray dosages of 1 mGy do not induce sudden severe cellular harm. Nevertheless, stochastic effects of ionizing irradiation, such as mutations, do not have a dosage threshold and always represent the potential danger of alterations to cells and cellular components, especially the DNA. This could lead to undefined mutations inducing genetic drift, in the worst case to the loss of a mutant line. We therefore strongly recommend minimizing "transportation stress," in particular by irradiation exposure, to keep its potential consequences in mind, and to standardize shipping procedures.

ZusammenfassungZucht und Haltung immundefizienter Labortiere wurden bislang nicht als Tierversuch gewertet, soweit die Tiere mit an Sicherheit grenzender Wahrscheinlichkeit nicht aufgrund des Immundefekts leiden müssen. Das aufgrund des Wahrscheinlichkeitsmaßstabs hinzunehmende Restrisiko, insbesondere durch äußere Ereignisse hat sich in der versuchstierkundlichen Praxis nicht realisiert. Trotzdem wertet die EU-Kommission nunmehr Zucht und Haltung immundefizienter Labortiere grundsätzlich als Tierversuch – mit erheblichem administrativem Mehraufwand in den mit Tieren forschenden Wissenschaften und der behördlichen Praxis. Im Folgenden wird aus versuchstierkundlicher und rechtlicher Perspektive festgestellt, dass diese Neubewertung der Kommission weder sachgemäß noch verhältnismäßig ist.

Article 23(2) of the European Union Directive 2010/63/EU, which regulates welfare provisions for animals used for scientific purposes, requires that staff involved in the care and use of animals for scientific purposes be adequately educated and trained before they undertake any such work. However, the nature and extent of such training is not stipulated in the Directive. To facilitate Member States in fulfilling their education and training obligations, the European Commission developed a common Education and Training Framework, which was endorsed by the Member States Competent Authorities. An Education & Training Platform for Laboratory Animal Science (ETPLAS) Working Group was recently established to develop further guidance to the Learning Outcomes in the Framework, with the objective to clarify the levels of knowledge and understanding required by trainees, and to provide the criteria by which these Learning Outcomes should be assessed. Using the Framework document as a starting point, assessment criteria for the Learning Outcomes of the modules required for Function A persons (carrying out procedures on animals) for rats, mice and zebrafish were created with sufficient detail to enable trainees, providers and assessors to appreciate the level of knowledge, understanding and skills required to pass each module. Adoption and utilization of this document by training providers and accrediting or approving bodies will harmonize introductory education and training for those involved in the care and use of animals for scientific purposes within the European Union, promote mutual recognition of training within and between Member States and therefore free movement of personnel.

The health care system of Germany approved a marginal number of telemedicine applications for reimbursement. However Teleradiology and Telestroke networks are well adopted in Germanys hospitals. Heart failure is the main indication for remote patient management and therefore a field of intense research in Germany. In almost every medical field telemedicine undergoes research and development. The lack of evidence in the field of telemedicine is due to the complexity of these interventions. Evaluation needs more resources and different trial designs compared to RCTs in pharmaceutical trials. A large scale funding program (Innovationsfonds) in Germany encompasses telemedicine and outcomes research. Mobile health apps are a new and expanding field in healthcare. Health apps are potentially beneficial in many health care areas, although there is little scientific evidence available. From a legal perspective data protection and privacy and the classification of health apps as medical devices are important topics.

Background In atrial fibrillation–associated stroke, conflicting data exist regarding association between therapeutic vitamin K-antagonist anticoagulation (International Normalized Ratio 2–3) and early death and functional outcome, and few data exist relating to late outcome in ischemic and haemorrhagic atrial fibrillation–stroke. Aim We performed a systematic review and meta-analysis of oral anticoagulation at stroke onset, death and functional outcome. Methods We performed a systematic review, searching multiple sources. Studies were included if outcomes in atrial fibrillation–associated stroke were reported stratified by pre-stroke antithrombotic status, with documented International Normalized Ratio at onset. Outcomes were survival and good functional outcome (modified Rankin score 0–2) at discharge/30 days, and at one-year. Results Of eight studies (3552 patients) in ischemic stroke, International Normalized Ratio ≥ 2 compared with other treatments (International Normalized Ratio < 2, antiplatelet, or no anti-thrombotic) was associated with good outcome [pooled odds ratio 1·9 (95% confidence interval) 1·5–2·5, P < 0·001] and improved survival at 30 days discharge (pooled odds ratio for death 0·4, confidence interval 0·2–0·5, P < 0·001). The net benefit remained after inclusion of haemorrhagic stroke (odds ratio for good outcome 1·89, confidence interval 1·45–2·46, P < 0·001). At one-year, improved functional outcome for International Normalized Ratio ≥ 2 (pooled odds ratio 1·7, confidence interval 1·0–2·7, P = 0·04) and survival (odds ratio for death 0·5, confidence interval 0·4–0·8, P = 0·001) were also observed. Conclusions Therapeutic International Normalized Ratio at stroke onset was associated with early and late improved survival and functional recovery suggesting sustained benefit for warfarin anticoagulation for stroke outcome in atrial fibrillation patients. Long-term outcome data following stroke in patients taking new oral anticoagulants is required.

Title: Genetically modified animals, in particular mice, are unique mutants with a major scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to protect those lines against loss or to archive them. Prerequisite to take a mutant line out of a breeding nucleus is the cryopreservation of a sufficient number of samples and a powerful quality assessment. Background: Several common protocols to cryopreserve spermatozoa and subsequently to forward these to an in vitro-fertilization under standardized conditions are published using donors of a strictly limited age-span. In practice, several limitations happen frequently for those approaches, e.g. the availability of genetically modified males, in particular if a line is in an experimental use or the mutation leads to restrictions. Female donors are often received from external sources and are subsequently not available before weaning. They are subject of several delays to induce an early superovulation. The aim of this report is to study the suitable age span of sperm and oocyte donors. Methods and Findings: A total of 441 GM sperm donors and the production of wild type oocytes with the genetic backgrounds BALB/c, C57BL/6N, and DBA/2 were investigated. The quality was assessed with simple, but purposive scores. Polynomial statistical analyses investigating increasing and decreasing yields show that the age of sperm-donors can be extended to ten months; the suitable age of oocyte donors is strain specific up to twelve weeks. Conclusions: The extended age spans demonstrated allow a more flexible use of donors and contribute subsequently to the 3Rs.

Genetically modified animals are unique models with enormous scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if a sufficient number of samples have been cryopreserved. To maintain the opportunity to recover a line, it is mandatory to assess the quality of the cryopreserved samples and to assure safe long-term storage conditions. Here, we investigated the revitalization rate of cryopreserved pre-implantation embryos stored in-house up to 158 months, of imported (and shipped) embryos, and of embryos received after in vitro fertilization. The storage period did not affect the revitalization rate, whereas the recovery of imported embryos was significantly reduced, possibly due to shipment conditions. The genotypes of genetically modified pups received following embryo-transfer were slightly smaller than expected by Mendelian laws. Intensive investigations of the hygienic state of the cryopreserved samples and the equipment used never showed microbiological contamination of a sample within a cryo-tube. However, environmental organisms were found frequently in the permanent freezers and dry shippers used. Since such contamination cannot be completely excluded and an embryo-transfer might not lead in all cases to a secure rederivation, foster mothers and revitalized pups should be housed in an intermediate facility and their health assessed before introducing them into the target facility.

Connexins are the structural units of gap junctions, structures allowing interchanging of information between the adjacent cells. Connexin43 (Cx43) is the most abundant gap junction protein. Cx43 can be degraded by lysosome- and proteasome-mediated processes upon internalisation of the entire structure. Only little is known about the role of phosphorylation during the gap junction degradation. In Cx43, a protein containing 14 amino acids susceptible to be phosphorylated, amino acids S279 and S282 are phosphorylated upon epidermal growth factor (EGF) treatment by erk1/2 MAP kinases. Here, we show that the wild-type Cx43 protein, as well as HeLa cells expressing the mutated Cx43 proteins S279A, S282A, and S279A/S282A, is mainly located at the plasma membrane. However, the protein stability is not altered in the isolated single mutants, whereas the double mutant S279A/S282A is strongly degradation impaired upon EGF treatment. This effect is not due to the decreased Cx43 internalisation, but seems to be related to a reduced ubiquitination.

In order to improve access to critical patient data in case of emergency, many countries have begun or intend to implement emergency datasets. In Germany, the German Medical Association developed a medical emergency dataset (MED), which provides the possibility to store information on prior diagnoses, medications, allergies and other emergency-relevant information on the German Electronic Health Card.The aim of the study is to evaluate how the MED can be used internationally.A total of 64 paper-based emergency data sets were completed by primary care physicians in Germany, and were then evaluated by German clinicians, emergency physicians, and paramedics on the basis of fictitious emergency scenarios. Thirty randomly selected MEDs were then translated into English and will be evaluated by international emergency physicians and paramedics.In Germany, clinicians, emergency physicians and paramedics rated the emergency data set as very useful or useful in more than 70% of the reviewed cases. The international evaluation will start in September 2016, so these results are pending at this time.The first study results from Germany indicate high potential benefits of the emergency data set in real patient care situations. The subsequent tests will show whether the MED is also suitable for international use.

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).

Generation and cryopreservation of transgenic mice depend on reliable and continuous production of pre-implantation embryos. To suppress circannual and circadian rhythms driving the physiological and sexual behaviour of free living animals, laboratory animals are housed under standardized conditions. It remains to be elucidated if the artificial climate can cover all environmental effects. Here, we report that the humidity in an animal facility affects the embryo yield. The weather at the location of the facility, especially the temperature, influences the climate within an animal facility; weather peaks are obviously covered in part only, even if the facility is equipped with a powerful air-conditioning supply. Subsequently, external weather changes interact with the environment within the facility, influencing the production of embryos. Furthermore, noise and/or vibrations as generated by construction works, negatively affect the embryo yield.

Background: Stroke prevention with oral anticoagulation is effective in patients with atrial fibrillation. An additional beneficial effect of oral anticoagulation prior to the event on stroke severity and prognosis was suggested in ischemic strokes. We tested whether this benefit is preserved after inclusion of hemorrhagic strokes and adjustment for prestroke living conditions. Methods: Data were used from a prospective hospital-based intervention trial evaluating quality of care and outcome in ten district hospitals. All ischemic and hemorrhagic stroke patients with atrial fibrillation were included. We analyzed separate multivariable regression models to identify factors associated with prescription of oral anticoagulation before stroke and to investigate the independent effect of anticoagulation on admission stroke severity, 3-month mortality and functional outcome. Results: The analysis comprised 804 (718 ischemic, 86 hemorrhagic) stroke patients admitted between July 2003 and March 2005. Males, patients aged 65–84, living independently, with diabetes, previous cerebrovascular event or additional high cardioembolic risks were more likely to receive oral anticoagulation before admission. Admission international normalized ratio (INR) between 2 and 3 (OR 0.35, 95% CI: 0.17–0.71) or higher (OR 0.32, 95% CI: 0.11–0.92) was associated with less severe strokes (including hemorrhagic strokes) compared with INR 3) at 3 months (OR 0.54, 95% CI: 0.36–0.84, and OR 0.70, 95% CI: 0.47–1.06). After adjustment for stroke severity, anticoagulation had no additional effect on mortality and functional outcome. Conclusions: The beneficial effect of prestroke anticoagulation on stroke outcome related to the reduced stroke severity is not offset by adverse effects in hemorrhagic stokes.

We generated mice expressing a full-length Mpl transgene under the control of a 2-kb Mpl promoter in an Mpl−/− background, effectively obtaining mice that express full-length Mpl in the absence of other Mpl isoforms. These mice developed thrombocytosis with platelet levels approximately 5-fold higher than wild-type controls and markedly increased megakaryocyte numbers. The reintroduction of one wild-type Mpl allele restored normal platelet counts. We excluded the deletion of Mpl-tr, a dominant-negative isoform, as the underlying molecular cause for thrombocytosis. Instead, we found that transgene expression driven by the 2-kb Mpl promoter fragment was decreased during late megakaryocyte maturation, resulting in strongly diminished Mpl protein expression in platelets. Because platelets exert a negative feedback on thrombopoiesis by binding and consuming Tpo in the circulation through Mpl, we propose that the severe reduction of Mpl protein in platelets in Mpl-transgenic Mpl−/− mice shifts the equilibrium of this feedback loop, resulting in markedly elevated levels of megakaryocytes and platelets at steady state. Although the mechanism causing decreased expression of Mpl protein in platelets from patients with myeloproliferative disorders differs from this transgenic model, our results suggest that lowering Mpl protein in platelets could contribute to raising the platelet count.

Background: Oral anticoagulation is highly effective for secondary prevention of cardioembolic strokes in patients with atrial fibrillation (AF). There are no studies investigating timing and complications of different strategies for initiation of oral anticoagulation after acute stroke or transient ischaemic attack (TIA). Methods: Patients of ten community hospitals participating in the prospective evaluation of medical effects of the Telemedical Project for Integrative Stroke Care (TEMPiS) were included. This observational evaluation was restricted to ischaemic stroke or TIA patients with AF who were started on Phenprocoumon treatment during in-hospital stay. Antithrombotic co-medication was dichotomized in heparin bridging (weight or partial thromboplastin time-adjusted heparin) or conventional treatment (antiplatelets and/or low-dose heparin or nil). Besides treatment-relevant extracranial bleeding, major complications were documented according to the European Atrial Fibrillation Trial definitions including vascular death, ischaemic or haemorrhagic stroke, systemic embolism, and myocardial infarction. Results: Between July 2003 and March 2005, 4,082 ischaemic stroke or TIA patients were admitted. AF was recorded in 961 patients (23.5%), of whom 376 (39.1%) received oral anticoagulation. In 229 of these patients oral anticoagulation was started in hospital, 150 (65.5%) with heparin bridging and 79 (34.5%) with conventional treatment. Patients with heparin bridging were younger, and had a longer in-hospital stay after adjustment for potential confounders (p = 0.01). Major complications were infrequent in both groups (2.0 vs. 2.5%; p = 1.0) as well as extracranial bleeding (3.3 vs. 1.2%; p = 0.43). Conclusions: Initiation of oral anticoagulation after acute ischaemic stroke yielded low complication rates independent of antithrombotic co-medication. Heparin bridging was associated with a longer stay in acute care hospitals.

Background: State-of-the-art stroke management requires neurological expertise for the recognition of complex cerebrovascular syndromes or stroke-mimicking symptoms and initiation of proven acute therapies. Many community hospitals struggle to fulfill these premises particularly at evening/nighttimes or weekends. Telemedicine can improve that situation by offering rapid access to neurological expertise, but it has not been shown to what extent it is used beyond working times. Methods: The Telemedical Project for Integrated Stroke Care is a telemedical network of 2 stroke centers and 12 regional general hospitals with newly established stroke wards in Bavaria. This analysis comprises all teleconsultations from 1st February 2003 to 15th December 2006. The consultations were prospectively documented and categorized according to predefined indications and direct impact on clinical decisions. The teleconsultations were analyzed concerning whether they were requested during regular working time or during off-time (at evening/nighttimes or weekends). Results: A total of 10,239 teleconsultations were carried out in 8,326 patients. The 6,679 patients with cerebrovascular diagnosis comprised 51% of all admitted stroke cases between 2003 and 2006. During off-time 6,306 consultations (62%) were requested; 1,598 teleconsultations yielded nonstroke diagnoses, with 68% beyond working hours. Of all presented stroke patients 567 (8.5%) received systemic thrombolysis, with 58% off-time. Interhospital transports were initiated in 1,050 patients (10.5% of all), mainly for specific diagnostic workup or interventional treatments. Sixty percent of these transfers were launched off-time. Conclusions: The majority of teleconsultations were requested beyond normal working times and a significant proportion had an immediate impact on clinical decisions.

Background and Purpose— Basilar artery occlusion (BAO) is a stroke subtype with poor prognosis, but recanalizing therapies have been reported to be effective. We investigated whether initial admission to telemedically linked general hospitals with subsequent stroke-center transfer is related to poorer outcome than direct admission to stroke centers. Methods— All BAO cases of 3 stroke centers in Munich and 1 center in Regensburg between March 1, 2003 and December 31, 2004 were included, either if patients were directly admitted to stroke centers (n=23) or had initial admission to general hospitals of the telemedical network for integrative stroke care (TEMPiS) and secondary transfer to stroke centers (n=16). BAO was defined as angiographically (CTA, MRI or conventional angiography) confirmed occlusion of the basilar artery. Baseline parameters and therapeutic procedures were recorded. One-year follow-up was conducted prospectively. Results— Differences in baseline parameters were not statistically significant. Time from onset to first angiography was significantly longer in patients with secondary transfer (mean: 355±93 minutes versus 222±198 minutes; P P P P =0.02) were living at home after 1 year. Conclusions— BAO patients who were admitted primarily to community hospitals had a worse prognosis. Patients with typical symptoms should have direct access to stroke centers, or may need bridging therapies.

In order to improve access to pre-existing patient information in case of emergency, the German Electronic Health Card (EHC) is supposed to hold emergency data. As a basis, the German Medical Association developed an emergency data set, which provides the possibility to store information on prior diagnoses, medications, allergies and other emergency-relevant information.One main objective of the study is to evaluate the usefulness of the emergency data in specific emergency situations.Within a two-phase exploratory study, a total of 64 paper-based emergency data sets were completed by primary care physicians, and then were evaluated by clinicians, emergency physicians, and paramedics.Clinicians, emergency physicians as well as paramedics rated the emergency data set in more than 70% of the reviewed cases as very useful or useful. The greatest benefit was attributed to the information on diagnoses and medication.The implementation of an emergency data on the EHC has the potential to improve safety, quality and efficiency of emergency care.

Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance.

In an acute emergency, the knowledge of the patient's medical history, allergies, implants, and medication can be crucial. ICD-10 code to document medical diagnoses alone often does not contain enough information. Our comparison of 388 documented diagnoses (ICD-10 codes as well as free text) showed that almost 20% of all coded ICD-10 codes contained less information than the documented free text. Thus, if using ICD-10 codes, free text diagnoses must be a necessary item in the upcoming German Medical Emergency DataSet.

Telemedical networks are a new approach to improve stroke care in community settings. We aimed to assess the effects of a stroke network with telemedical support in Germany on quality of care, according to acute processes and long-term outcome.Five community hospitals without pre-existing specialised stroke care were included in a network with telemedical support by two academic hospitals. In a non-randomised, open intervention study, five community hospitals without specialised stroke care served as the control group, matched individually to the network hospitals by predefined characteristics. Stroke patients admitted consecutively to one of the participating hospitals between July 7, 2003, and March 31, 2005, were included in the study. Patients in network and control hospitals were assessed in the same manner and were followed up for vital status, living situation, and disability at 3 months. Poor outcome was defined by death, institutional care, or disability (Barthel index60 or modified Rankin scale3). Predefined indicators for quality of acute stroke care were achieved.A total of 5696 patients with a sudden, non-convulsive loss of neurological function who were diagnosed with having suspected stroke were admitted to the ten hospitals participating in the study. After exclusion, 3122 were included in the final analysis, of whom 1971 (63%) were treated in the network hospitals. All indicators related to quality of acute stroke care were more commonly met in the network than in the control hospitals. After 3 months, 44% of patients treated in network hospitals versus 54% treated in control hospitals had a poor outcome (p0.0001). In multivariate regression analysis, treatment in network hospitals independently reduced the probability of a poor outcome (odds ratio 0.62, 95% CI 0.52-0.74; p0.0001).Telemedical networks with academic stroke centres offer new and innovative approaches to improve acute stroke care at community level for stroke patients living in non-urban areas.

A total of 70,629 predominantly transgenic mouse embryos prepared from 9,727 pregnant female donors were cryopreserved using a method leading to a high revitalisation rate. Against loss, 125 mutant mouse lines were protected. An average of 7.26 embryos (eight-cell embryos) per pregnant donor was received. To reduce the number of animals required as embryo donors, a special breeding exclusively for cryopreservation was omitted if possible and subsequently the number of animals used for freezing was reduced remarkably. The advantage of this strategy is that (mutant) mouse lines out of current use do not have to be kept in a breeding nucleus. In parallel, this procedure leads to rederivation and improves the export of mice to other facilities. The cryopreservation of these 125 mutant lines keeps the potential to save approximately 20,000 laboratory mice per year to be bred if they were kept in a breeding stock. This is a major contribution to the “3R” requirements developed by Russel and Burch to reduce the number of laboratory animals.

Background: Providing stroke unit treatment for all stroke patients is a cross-national goal as stated in the WHO Helsingborg Declaration. In order to achieve specialized stroke care for a large area, two stroke centers and 12 community hospitals established an integrative stroke network. This evaluation was performed to analyze achieved advances in stroke management. Methods: Core network elements are: (1) establishing stroke wards in all hospitals; (2) continuous training in stroke treatment; (3) telemedicine service staffed by a 24 h/day ‘strokologist’ with capability for high-speed videoconferencing and transfer of CT/MRI images. Data were prospectively documented in the databank of the telestroke service, in the Bavarian Stroke Registry and in the controlling departments. Results: In 2003, 4,179 stroke patients were admitted to the regional network hospitals. Between February 2003 and January 2004 a total of 2,182 teleconsultations were conducted. 250 teleconsultations yielded a nonvascular diagnosis. Indicators for stroke management quality improved compared with other hospitals without stroke unit: the frequency of CT/MRI within 3 h was 59% compared to 46%, frequency of speech therapy 36% (21%), and of occupational therapy 38% (12%). Eighty-six (2.1%) of the patients received systemic thrombolysis compared to 10 patients in the preceding year. Mean length of in-hospital stay decreased from 12.4 in 2002 to 9.7 days in 2003. Conclusions: This stroke network concept leads to a substantial improvement of stroke management. Telemedicine contributes to an early etiological assessment and fills the gap of specialized stroke expertise in neurologically underserved areas.

Neurodegenerative diseases often result in neuronal cell death, but the molecular mechanisms responsible are not fully understood. The expression and activation by phosphorylation of the c-Jun transcription factor plays an important role for the fate of affected neurons in response to injury. c-Jun is phosphorylated at serines 63 and 73 by the c-Jun N-terminal kinases and c-Jun N-terminal phosphorylation augments the transcriptional activity of c-Jun. Two approaches in neurodegeneration were investigated: The transection of the medial forebrain bundle to study neuronal cell body response in the derived neuronal populations of the substantia nigra pars compacta (SNC). This model of central axotomy leads as a long-term reaction to degeneration of the affected SNC neurons. A central component of the axotomy-induced alterations leading to neuronal degeneration is the rapid induction, lasting expression and activation of the c-Jun transcription factor. The focal cerebral ischemia, induced by occlusion of the arteria cerebri media and the subsequent reperfusion, serves as a suitable in vivo model for stroke. Also, ischemia leads to upregulation and activation of c-Jun and its target genes. Here the key role of c-Jun for the fate of neurons following degeneration is discussed with data received from experiments performed in Manfred Zimmermann's department investigating the effects of c-Jun on its target genes and on factors influencing c-Jun expression and activation.

Naïve CD4+ T cells differentiate into effector T helper 1 (Th1) or Th2 cells, which are classified by their specific set of cytokines. Here we demonstrate that loss of JunB in in vitro polarized Th2 cells led to a dysregulated expression of the Th2-specific cytokines IL-4 and IL-5. These cells produce IFN-gamma and express T-bet, the key regulator of Th1 cells. In line with the essential role of Th2 cells in the pathogenesis of allergic asthma, mice with JunB-deficient CD4+ T cells exhibited an impaired allergen-induced airway inflammation. This study demonstrates novel functions of JunB in the development of Th2 effector cells, for a normal Th2 cytokine expression pattern and for a complete Th2-dependent immune response in mice.

At weaning, milk producing mammary epithelial cells undergo apoptosis and are removed by phagocytosis. Here, we show that mouse mammary gland involution is associated with mitochondrial cytochrome c release and processing of numerous caspases, including caspase-1, -3, -7, -8 and -9. Induction of caspase-3-like activity paralleled cleavage of poly-(ADP--ribose) polymerase. Dexamethasone inhibited processing of caspase-3, -7 and -8 and apoptosis, but had no effect on caspase-1 accumulation and cytochrome c release. In Bcl-2 transgenic animals, cytochrome c release, caspase activation and apoptosis were impaired. Thus, the pro-apoptotic signaling pathway in mammary epithelial cells during involution involves the release of cytochrome c and activation of caspases. It is inhibited by Bcl-2 at the mitochondrial level and by dexamethasone at a post-mitochondrial level.

The immunophilin ligand FK506 (Tacrolimus) is used for prevention of graft rejection following organ transplantation. FK506 is a high-affinity ligand for FK506-binding proteins, an immunophilin subgroup of peptidyl-prolyl-cis/trans-rotamases abundant in the mammalian brain. Here, we demonstrate that FK506 is a potent survival factor that prevents neuronal cell death following axotomy of central intrinsic neurons. Administration of FK506 (2 mg/kg, s.c., per day for two days pre-axotomy and for up to eight days post-axotomy) effectively delayed and reduced the death of axotomized neurons in the substantia nigra pars compacta following transection of the medial forebrain bundle. In saline-treated controls, 75%, 89% and 92% of nigral neurons died after 25, 50 and 60 days post-axotomy, respectively. In contrast, application of FK506 resulted in survival of 46%, 44% and 28% of the axotomized nigral neurons, and the majority of these surviving neurons showed continuous expression of tyrosine hydroxylase, the pacemaker enzyme for dopamine synthesis. Moreover, FK506 significantly reduced the expression of the inducible transcription factor c-Jun and its N-terminal phosphorylation and prevented the axotomy-induced suppression of the constitutive transcription factor ATF-2 in neurons of the substantia nigra and mammillary body. The latter is also axotomized by the coincident transection of the mammillothalamic tract, but the mammillary neurons survive the axotomy. In contradistinction to FK506, the non-immunosuppressive FK506-binding protein ligand GPI-1046 (25 or 12.5 mg/kg, applied once or twice per day for two days pre-axotomy and for eight days post-axotomy) was completely ineffective for all these parameters investigated. Finally, FK506, but not GPI-1046, impressively accelerated the recovery from surgery. Our data provide the first evidence that FK506 acts as a neuroprotective molecule that rescues axotomized otherwise degenerating central intrinsic neurons in the adult mammalian brain by mechanisms that interfere with the transcriptional program of the axotomy-induced cell body response, such as activating transcription factor-2 suppression and c-Jun expression and phosphorylation.

We have generated transgenic mice carrying the URR of the human papillomavirus type 11 ligated in front of the Escherichia coli beta-galactosidase coding region sequence. Using X-Gal staining to demonstrate beta-galactosidase production, we observed a hair-specific transcription of the reporter gene. This transcription was limited to the epithelial cells of the hair bulge region. The transgene was developmentally regulated, as no LacZ staining was demonstrated during embryogenesis and specific staining was first observed after birth. Surprisingly, dexamethasone and ultraviolet B, but not phorbol myristate acetate or progesterone treatment of the animals resulted in an increase in number and intensity of hair follicles expressing the reporter gene.

Expression of the apoptosis-inhibitory protein Bcl-2 has frequently been detected in human cancer including mammary carcinoma. The functional significance of its expression has been well established in experimental tumors of the lymphoid system, however, remains to be elucidated for epithelial tumors. In order to assess the role of Bcl-2 in mammary tumorigenesis we have generated WAP-bcl-2 transgenic mice. The strong overexpression of Bcl-2 in lactating mammary glands was preserved during early postlactational involution and apoptosis of alveolar epithelial cells was prevented without influencing the dedifferentiation of the milk-producing epithelium. Although Bcl-2 overexpression was not sufficient to induce spontaneous tumors it, however, led to an accelerated development of MMTV myc transgene-induced mammary tumors. In the mammary glands of MMTV myc transgenic mice, a high proportion of apoptotic cells was detected which was significantly reduced in the mammary glands of WAP-bcl-2/ MMTV myc double transgenic mice. Taken together, these results suggest that Bcl-2 contributes to mammary tumorigenesis by inhibiting apoptosis.

The aim of the study was to evaluate the association of the body mass index (BMI) with mortality and with non-fatal functional outcome in patients with acute stroke or transient ischaemic attack (TIA). Obesity is an established risk factors in primary cardiovascular disease prevention including stroke. The impact of overweight in patients with stroke or TIA on secondary fatal and non-fatal functional outcomes is less well established.Data from 4428 patients with acute stroke or transient ischaemic attack (TIA) from the Telemedical Project for Integrative Stroke Care (TEMPiS) were studied in this post hoc analysis. The body mass index was available in 1521 patients. Patients were categorized as underweight (BMI18.5), normal (BMI 18.5 to25) overweight (BMI 25 to30), obesity (BMI 30 to35), advanced obesity (BMI ≥35 all kg/m(2)), and no body weight assessed. Outcome measures after 30 months were all-cause mortality and non-fatal outcomes: recurrent stroke, need for institutional care, and functional impairment (Barthel index60, modified Rankin score3). Mortality risk was lower in overweight patients [hazard ratio (HR): 0.69, 95% confidence interval (CI): 0.56-0.86) and lowest in obese (HR: 0.50, 95% CI: 0.35-0.71) and very obese patients (HR: 0.36, 95% CI: 0.20-0.66] compared with normal BMI. Functional, non-fatal outcomes, and recurrent stroke followed the same inverse pattern: underweight patients had the worst outcomes but obese patients had better outcomes than patients with normal BMI (all P0.01). After adjustment for multiple confounding factors, obese patients had a lower risk of the combined endpoints of death or institutional care (OR: 0.60, 95% CI: 0.38-0.92), death or high dependency (OR: 0.60, 95% CI: 0.39-0.91) and death or recurrent stroke (OR: 0.56, 95% CI: 0.37-0.86). Mortality was significantly lower in obese patients (all BMI30 kg/m(2)) than patients with normal weight (HR: 0.70; 95% CI: 0.50-0.98). Underweight patients had consistently the highest risks for all endpoints.Overweight and obese patients with stroke or TIA have better survival and better combined outcomes of survival and non-fatal functional status than patients with the BMI25 kg/m(2).

The number of genetically modified mice is increasing rapidly. Several limitations when working with these animals are to be considered: small colonies, the continued danger of loss, often a limited breeding-success, the need to keep those mutants in stock, difficult and costly import-procedures, and also a major (scientific) value of those mutants often available only with major restrictions. To gather relevant information about all active and archived genetically modified mouse lines available in-house (>1.500) and to deal with a unique resource for several, quite different purposes, a data base was developed enabling optimum knowledge management and easy access. The data base covers also legal restraints and is being linked with the institutional publication repository. To identify the lines available detailed information is provided for each line, as the international designation, a short name, the characterization/description, and the genetic modification including the technique used therefore. The origin of the mutation (gene-ID# and donor organism), the origin of regulatory elements and their donors are listed as well as the genetic background, back-cross generation, phenotype, possible publications, keywords, and some in-house information. Also aspects of animal welfare, obligations to record genetically modified organisms, and technology transfer are displayed; the latter to make licenses possible (if legally permitted). Material transfer agreements, patents, or legal restrictions are listed. This data base helps to avoid double-imports, saves animals and costs since a redundant generation or import can be omitted. However, this is a contribution to the 3R principles developed by Russell and Burch.

Introduction: Stress and environmental perturbations influence postnatal brain development and may account for the high disability rates of preterm survivors following intensive care treatment. This study aims to investigate the impact of early environmental factors on the monoaminergic neurotransmitter system in the developing rat brain by using an innovative neonatal stress model. Materials and methods: After birth, in the experimental groups newborn rats were separated from their mothers and exposed to different stressful stimuli four times a day on day P0 to P6 for 10 min each. To mimic intensive care treatment, the stress protocol applied environmental factors like bright light, noise, and low temperature alternating with pain and handling stress at day- and night-time in a varying sequence. The non-stressed control mothers and litters were left completely undisturbed until sacrificing on day P7 or P20. Results: Brains of stressed animals revealed significantly higher levels of norepinephrine (NE) and dopamine (DA) as determined by HPLC-ED and electrochemical detection at day P7 as compared to controls. When returned to their mothers’ undisturbed care, juvenile rats at day P20 still showed higher (yet statistically not significant) concentrations of NE and DA in brain. The stressed animals gained less weight with significantly lower body weights at day P7 compared to controls. Their mothers developed various forms of stressed behaviour. Conclusions: A novel animal model for postnatal intensive care stress was established leading to changes in brain monoamine levels of newborn rats, while undisturbed maternal care seems to moderate the stress effects subsequently.

Activation of mature lymphocytes requires in addition to the TCR contact with the corresponding antigen the binding of the CD8 or CD4 co-receptors to MHC class I or class II proteins respectively. To investigate the contribution of the CD8-class I interaction to the elimination of autoreactive T cells during negative selection in the thymus we generated two types of transgenic mice. One set expressed a modified Kb molecule which contained a human HLA-A2 alpha 3 domain, thereby missing the binding residues for the murine CD8 molecules. The second set of mice expressed an anti-Kb specific TCR. Both lines were crossed and in the resulting double transgenic mice the development of Kb-reactive T cells was followed with an anti-clonotypic antibody. Surprisingly, efficient clonal deletion in the thymus was still observed, although the reduced CD8-class I adhesion abrogated effector functions in vivo and in vitro. These results imply that even T cells with intermediate affinity for self are negatively selected in the thymus despite the fact that they are not able to react against self antigens in the periphery. Thus a safety window is created which decreases the risk of autoaggression.