Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

[Cost analysis of telemedical treatment of stroke]. / Kostenanalyse telemedizinischer Schlaganfallbehandlung.

BACKGROUND: Telemedicine-enabled stroke networks increase the probability of a good clinical outcome. There is a shortage of evidence about the effects of this new approach on costs for inpatient care and nursing care. METHODS: We analysed health insurance and nursing care fund data of a statutory health insurance company (AOK Bayern). Data from stroke patients initially treated in a TeleStroke network (TEMPiS - telemedical project for integrative stroke care) between community hospitals and academic stroke centres were compared to data of matched hospitals without specialised stroke care and telemedical support. Costs for nursing care were obtained over a 30-month period after the initial stroke. To rule out pre-existing differences between network and control hospitals, costs of stroke care were also analysed during a time period before network implementation. FINDINGS: 1 277 patients (767 in intervention, 510 in control hospitals) were analysed in the post-implementation period. An increased proportion of patients treated in intervention hospitals had a favourable outcome concerning the level of required nursing care. Patients in intervention hospitals had higher costs for acute inpatient care (5 309 € vs. 4 901 €, p=0.04), but lower nursing care fund costs (3 946 € vs. 5 132 €; p=0.04). There was no difference in relation to absolute total costs obtained in the post-implementation period. However, nursing care costs per survived year were significantly lower in intervention hospitals (1 953 € vs. 2 635 €; p=0.005). No significant differences were found in the pre-implementation period. CONCLUSIONS: Considering both health insurance and nursing care fund costs, the incremental costs for TeleStroke network care in hospitals are compensated by savings in outpatient care.

Levels of peripheral T cell tolerance induced by different doses of tolerogen.

Antigen-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. A liver-specific inducible promoter was used to drive the expression of the major histocompatibility complex antigen Kb in transgenic mice. Minute amounts of Kb, expressed exclusively on hepatocytes, induced tolerance by partial down-regulation of the T cell receptor (TCR) on the self-reactive CD8+ cells. Contact of these tolerant T cells with high concentrations of Kb after induction led to complete down-regulation of TCR. Thus, tolerant T cells are susceptible to further tolerogenic signals and reach different levels of tolerance depending on antigen dose.

[Acceptance of telemedicine for acute stroke care. The German project TEMPiS]. / Akzeptanz der Telemedizin in der akuten Schlaganfallversorgung. Das bayerische Projekt TEMPiS.

BACKGROUND: Telemedicine is increasingly used for acute stroke care, making neurological expertise available in nonspecialized hospitals. There are few data about telemedicine's acceptance by either medical staff or patients at treating hospitals. METHODS: Telemedicine's acceptance was evaluated in the Telemedical Project for Integrative Stroke Care (TEMPIS), a network of two stroke centers and 12 community hospitals in the German state of Bavaria; the grading of teleconsultation regarding video and audio quality, time consumption, and medical relevance was assessed in two periods, 2004 and 2007. Overall satisfaction with in-hospital treatment was compared between patients in telemedically-linked hospitals with specialized stroke care and patients treated in conventional community hospitals. With regard to sufficient follow-up rates, ratings were restricted to patients living at home without severe disability at 3 months after stroke. A second evaluation analyzed how the parameter "Telemedical assessment of patient" (36% of patients in TEMPIS hospitals) affected overall satisfaction. RESULTS: Respectively, 140 and 127 uses of telemedicine were assessed in the two evaluation periods. Video quality, time consumption, and medical relevance were graded "excellent" by over 50% in both periods. Audio quality was rated "excellent" by only 22% in the first period but 69% in the second. Excellent overall satisfaction was expressed significantly more frequently by patients at TEMPIS hospitals (total number 1044) than by those at control hospitals (total number 484; 56% vs 47% respectively, P<0.01). Patient consultation via telemedicine per se did not correlate with modified grading. CONCLUSIONS: Acceptance of telemedicine in acute stroke care was high and stable over a long period. This study suggests improved overall satisfaction with treatment in hospitals offering specialized care and linked via telemedicine. Clinical assessment via telemedicine had no major effect on satisfaction.

Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice.

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.

CD95 ligand (Fas-L/APO-1L) and tumor necrosis factor-related apoptosis-inducing ligand mediate ischemia-induced apoptosis in neurons.

Programmed cell death plays an important role in the neuronal degeneration after cerebral ischemia, but the underlying mechanisms are not fully understood. Here we examined, in vivo and in vitro, whether ischemia-induced neuronal death involves death-inducing ligand/receptor systems such as CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). After reversible middle cerebral artery occlusion in adult rats, both CD95 ligand and TRAIL were expressed in the apoptotic areas of the postischemic brain. Further recombinant CD95 ligand and TRAIL proteins induced apoptosis in primary neurons and neuron-like cells in vitro. The immunosuppressant FK506, which most effectively protects against ischemic neurodegeneration, prevented postischemic expression of these death-inducing ligands both in vivo and in vitro. FK506 also abolished phosphorylation, but not expression, of the c-Jun transcription factor involved in the transcriptional control of CD95 ligand. Most importantly, in lpr mice expressing dysfunctional CD95, reversible middle cerebral artery occlusion resulted in infarct volumes significantly smaller than those found in wild-type animals. These results suggest an involvement of CD95 ligand and TRAIL in the pathophysiology of postischemic neurodegeneration and offer alternative strategies for the treatment of cardiovascular brain disease.

Involvement of CD95/Apo1/Fas in cell death after myocardial ischemia.

BACKGROUND: The death of cardiac cells during ischemia and reperfusion is partially mediated by apoptosis, as seen, eg, in autopsy material of patients after acute myocardial infarction. METHODS AND RESULTS: To study the role of CD95/Fas/Apo1 for induction of postischemic cell death, we used an ischemia/reperfusion model of isolated rat and mouse hearts in Langendorff perfusion. In this model, caspase-dependent apoptosis occurred during postischemic reperfusion. Moreover, soluble CD95 ligand/Fas ligand was released by the postischemic hearts early after the onset of reperfusion. In addition, this ligand was synthesized de novo under these circumstances. Similar findings were observed for other "death-inducing" ligands, such as tumor necrosis factor (TNF)-alpha and TNF-related apoptosis-inducing ligand. In primary adult rat myocyte culture, hypoxia and reoxygenation caused a marked increase in sensitivity to the apoptotic effects of CD95 ligand. Isolated hearts from mice lacking functional CD95 (lpr) display marked reduction in cell death after ischemia and reperfusion compared with wild-type controls. CONCLUSIONS: These data suggest that CD95/Apo1/Fas is directly involved in cell death after myocardial ischemia. The CD95 system might thus represent a novel target for therapeutic prevention of postischemic cell death in the heart.

Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment.

Mouse mammary gland involution is associated with cytochrome c release and caspase activation.

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 Hazard Analysis Critical Control Point's (HACCP) concept as applied to some chemical, physical and microbiological contaminants of milk on dairy farms. A prototype.

Quality management on dairy farms becomes more and more important regarding the different areas of animal health, animal welfare and food safety. Monitoring animals, farm conditions and farm records can be extended with risk identification and risk management. The hazard analysis critical control point's system is useful as an on farm strategy to control the product as well as the production process on the areas of animal health, animal welfare and food safety. This article deals in detail with the question how to develop a qualitative method where risk can be defined as an interaction between probability and impact. Two parts of the production process (milk harvest and treatment of cows) where used as an example how to apply the hazard analysis critical control point's system on chemical, physical and microbiological contaminants of milk. Not just only by summarizing the different critical checkpoints for each area but also by giving them a precise judgement of probability and impact.

The human papillomavirus type 11 upstream regulatory region triggers hair-follicle-specific gene expression in transgenic mice.

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.

In vitro reconstitution of hnRNP particles.

The assembly of hnRNP-like particles was studied by in vitro reconstitution, UV-crosslinking and CsCl-equilibrium centrifugation. Using total nuclear protein and RNA extracts from HeLa cells for RNP reconstitution, RNP particles sedimenting with the same buoyant density of p = 1.4 g/cm3 as 'native' 40 S core hnRNPs were obtained. Under the stringent reconstitution conditions used, hnRNP complexes containing only the Cl-core hnRNP protein could be identified.

Analysis of guinea pig leukocyte antigens using interspecies T cell hybrids.

In the guinea pig system, there are no T lymphocyte tumors available. In addition, in this species the production of T cell lines in vitro for analysis of T cell markers or function proved to be difficult. As an alternative approach, guinea pig X mouse and guinea pig X rat T cell hybridomas were obtained by fusion of guinea pig T cells with HAT-sensitive mouse or rat thymomas. In ten fusions a total of 578 hybrids were produced and kept in culture for periods ranging from weeks up to several months. The highest yield was obtained when the Mb C 12 line was used as tumor parent. BW 5147 and the rat thymoma W/Fu(C58NT)D gave slightly lower fusion efficiencies. The yield depended also on the pretreatment of the parental cells. Activation by MLR proved to be most effective. A number of interspecies hybrids expressed guinea pig T cell differentiation antigens as detected by a cell ELISA. The positive hybrids were recloned several times and exhibited a stable expression of these markers, even after continuous culture for more than 2 months. Western blot analysis was used to confirm antigen expression at the protein level by comparing the hybrids with both parental cells. The hybrids expressed the proteins of the guinea pig lineage. No reaction was observed with the murine or rat thymomas. Therefore, the application of interspecies T cell hybrids provides an alternative for studies of guinea pig T cells. The cells are easy to grow and to clone and may be stored frozen in liquid nitrogen. The hybrids should permit analysis at the clonal level also for functional studies.

The immunophilin ligand FK506, but not GPI-1046, protects against neuronal death and inhibits c-Jun expression in the substantia nigra pars compacta following transection of the rat medial forebrain bundle.

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.

cDNA cloning of the constant region genes of the guinea pig alpha/beta T-cell receptor.

A lambda gt11 cDNA library was prepared from activated guinea pig T-lymphocyte blasts. cDNA clones coding for the alpha-chain and beta-chain of the constant region of the guinea pig T-cell receptor were isolated by means of crosshybridization using the corresponding mouse cDNA genes. The guinea pig and the corresponding mouse cDNA genes hybridized in Northern blots with mouse mRNA of the same size. Guinea pig T-cell receptor mRNA showed the same size like its mouse counterpart. In contrast to the alpha-chain clone, genomic Southern blot analysis showed that the identified beta-chain gene fragment undergoes genomic rearrangement during T-cell differentiation.

Production of antibodies to the constant region of the mouse T-cell-receptor beta-chain.

To generate antibodies to the constant region of the mouse T-cell-receptor beta chain, the corresponding cDNA clone 86T5 was subcloned into the bacterial expression vector pEX2. The induced hybrid protein consisting of a 30 kDa T-cell-receptor segment and a beta-galactosidase carrier moiety was used for immunization. In Western blots using lysates of a T-cell-receptor-positive T-cell hybrid, the antisera obtained reacted with a 42 kDa protein under reducing and a 85 kDa protein under nonreducing conditions. The antisera did not react in binding assays with intact T lymphocytes, i.e. membrane-associated T-cell-receptor protein.

MAP kinase phosphatase 1 is expressed and enhanced by FK506 in surviving mamillary, but not degenerating nigral neurons following axotomy.

The MAP kinase phosphatase 1 (MKP-1), a dual serine-threonine phosphatase, inactivates the MAP kinases ERK and JNK/SAPK which are involved in neuronal survival and neuronal cell death following injury and degenerative stimuli. We have studied by immunocytochemistry whether regulation of MKP-1 is part of the cell-body response following nerve fiber transection. The expression of MKP-1 was investigated in axotomized neurons of the corpus mamillaris (CMm) and substantia nigra pars compacta (SNC) following transection of the mamillo-thalamic tract (MT) and the medial forebrain bundle (MFB), respectively. In contrast to the surviving CMm neurons, the vast majority of SNC neurons undergoes cell death following axotomy. MKP-1 immunoreactivity which is absent in untreated adult rats, appeared in CMm neurons 24 h following MT transection, reached a maximum after 2 days and persisted in a substantial proportion of CMm neurons until 20 days, the end of observation period. In contradistinction, MKP-1 could not be detected in the SNC neurons. MKP-1 immunoreactivity was virtually restricted to the nuclei of neurons. Subcutaneous injection of the immunosuppressant FK506 that protects axotomized SNC neurons against neuronal cell death, enhanced the expression of MKP-1 in CMm, but failed to do so in SNC neurons. The selective expression of MKP-1 in CMm is the first finding on a different regulation of components in the stress kinase signal pathway in surviving vs. degenerating axotomized neurons.

FK506 prevents stroke-induced generation of ceramide and apoptosis signaling.

Ceramide is a key mediator of apoptosis during the cellular stress response which is also involved in stroke-induced death. Transient occlusion of the middle cerebral artery (MCA) in rats led to a strong generation of ceramide as measured in thalamus and entorhinal cortex of the ischemic brain tissue. Enhanced levels of ceramide may be involved in apoptosis signaling following stroke since exogenously added synthetic C2-ceramide increased expression of c-jun and the death-inducing ligands (DILs) CD95-L, TRAIL and TNF-alpha in neuroblastoma cells. DILs in turn mediated death via binding to their respective receptors as concluded from diminished apoptosis upon blocking of the common pathway by dominant negative FADD. C2-ceramide induced both necrosis and apoptosis in a concentration-dependent manner corresponding to the situation present in the ischemic brain. The immunosuppressant FK506 inhibited the release of ceramide, expression of CD95-L and apoptosis in an in vitro and in vivo model for ischemia/reperfusion. These data suggest that ceramide is a crucial initiator of death, e.g., by induction of DILs following stroke.

R1--systemic thrombolysis in German stroke units--the experience from the German Stroke data bank.

BACKGROUND: Systemic thrombolysis with tissue plasminogen activator (t-PA) for treatment of acute ischemic stroke was approved in Germany in 2000. Up to now, only data from single centers have been available for the study of the use of thrombolysis in a hospital-based approach outside controlled trials. We therefore sought to determine the frequency of application and complications as well as the patient outcome after t-PA treatment in clinical routine of specialized stroke centers in Germany. METHODS: Within the German Stroke Data Bank Collaboration, 6234 consecutive patients with ischemic stroke were prospectively documented in 20 stroke centers between 1998 and 1999. The patients were centrally followed via telephone interview after 3 months and 1 year to assess global functional outcome using the Modified Rankin Scale. RESULTS: 250 patients (4 %) received systemic t-PA treatment during the study period. The baseline characteristics of these patients were comparable to large clinical trials and phase IV studies. Symptomatic and asymptomatic parenchymal hemorrhage occurred in 22 patients (8.8 %) and was fatal in 3 patients. Follow-up data after 3 months were obtained in 82.4 % of all patients, of which 35 % had a favorable functional outcome (mRS /= 4) and 17 % had died. CONCLUSION: The results of our study agree with the assumption that thrombolytic therapy can be performed safely and effectively in daily clinical practice. Nevertheless, the small proportion of patients receiving thrombolysis even in specialized stroke centers calls for further improvement of acute stroke management in Germany.

Alcohol consumption in relation to food intake and smoking habits in the Dutch National Food Consumption Survey.

The interrelationships between alcohol consumption, energy and food intake and smoking habits were studied in 1145 men and 1171 women, aged 22-49 years, in the Dutch National Food Consumption Survey, in which a 48-h dietary record method was used. The aim of the study was to investigate the effects of alcohol consumption on dietary habits and smoking. A strong relationship between alcohol consumption and energy intake was found. The energy derived from alcohol was not compensated for by lower intake of other nutrients. There was no increase in Quetelet's index with increasing alcohol consumption, except for non-smoking men who were heavy drinking on midweek days. Possible explanations for this apparent lack of an overall effect of alcohol calories are discussed. Alcohol consumption was much higher on weekend days than on midweek days. No differences in nutrient intake were found between non-drinkers, moderate drinkers and heavy drinkers on midweek days. On weekend days, however, there was a slightly higher total fat and saturated fat intake in moderately drinking men. For women cholesterol intake was found to be higher in moderate and heavy drinkers. Finally, a strong positive relationship between alcohol consumption and smoking was observed. It is concluded that the observations with respect to energy and nutrient intake and smoking habits are not indicative of a healthier lifestyle in moderate alcohol users between 22 and 49 years of age. Consequently, the more favourable prognosis of moderate drinkers cannot be ascribed to a more healthy lifestyle.