In vitro culture of Mesocestoides corti metacestodes and isolation of immunomodulatory excretory-secretory products.

Cestode-mediated diseases hold the interesting feature of persisting metacestode larvae dwelling within the host tissues, in the midst of the immune response. Excretory-secretory (ES) products of the metacestode larval stage modulate the host immune response and modify the outcome of the disease. Therefore, isolation and analysis of axenic metacestode ES products are crucial to study their properties. Here, we report the development of a system for long-term in vitro cultivation of the metacestode of the parasitic cestode Mesocestoides corti (syn. Mesocestoides vogae). Although feeder cells and host serum supported the early growth of the parasite, long-term survival was not dependent on host serum or host-derived factors enabling the collection of parasite released products in serum-free medium. Functionally, these axenic ES products recapitulated M. corti tetrathyridia's ability to inhibit LPS-driven IL-12p70 secretion by dendritic cells. Thus, our new axenic culture system will simplify the identification and characterization of M. corti-derived immunomodulatory factors that will indirectly enable the identification and characterization of corresponding factors in the metacestode larvae of medically relevant cestodes such as Echinococcus multilocularis that are not yet amenable to serum-free cultivation.

Serological survey of Bartonella spp., Borrelia burgdorferi, Brucella spp., Coxiella burnetii, Francisella tularensis, Leptospira spp., Echinococcus, Hanta-, TBE- and XMR-virus infection in employees of two forestry enterprises in North Rhine-Westphalia, Germany, 2011-2013.

We initiated a survey to collect basic data on the frequency and regional distribution of various zoonoses in 722 employees of forestry enterprises in the German state of North Rhine-Westphalia (NRW) from 2011 to 2013. Exposures associated with seropositivity were identified to give insight into the possible risk factors for infection with each pathogen. 41.2% of participants were found to be seropositive for anti-Bartonella IgG, 30.6% for anti-Borrelia burgdorferi IgG, 14.2% for anti-Leptospira IgG, 6.5% for anti-Coxiella burnetii IgG, 6.0% for anti-Hantavirus IgG, 4.0% for anti-Francisella tularensis IgG, 3.4% for anti-TBE-virus IgG, 1.7% for anti-Echinococcus IgG, 0.0% for anti-Brucella IgG and anti-XMRV IgG. Participants seropositive for B. burgdorferi were 3.96 times more likely to be professional forestry workers (univariable analysis: OR 3.96; 95% CI 2.60-6.04; p<0.001); and participants seropositive for Hantavirus 3.72 times more likely (univariable analysis: OR 3.72; 95% CI 1.44-9.57; p=0.007). This study found a surprisingly high percentage of participants seropositive for anti-B. henselae IgG and for anti-F. tularensis IgG. The relatively high seroprevalence for anti-Leptospira IgG seen in this study could be related to living conditions rather than to exposure at work. No specific risk for exposure to C. burnetii and Echinococcus was identified, indicating that neither forestry workers nor office workers represent a risk population and that NRW is not a typical endemic area. Forestry workers appear to have higher risk for contact with B. burgdorferi-infected ticks and a regionally diverse risk for acquiring Hantavirus-infection. The regional epidemiology of zoonoses is without question of great importance for public health. Knowledge of the regional risk factors facilitates the development of efficient prevention strategies and the implementation of such prevention measures in a sustainable manner.

Cestode genomics - progress and prospects for advancing basic and applied aspects of flatworm biology.

Characterization of the first tapeworm genome, Echinococcus multilocularis, is now nearly complete, and genome assemblies of E. granulosus, Taenia solium and Hymenolepis microstoma are in advanced draft versions. These initiatives herald the beginning of a genomic era in cestodology and underpin a diverse set of research agendas targeting both basic and applied aspects of tapeworm biology. We discuss the progress in the genomics of these species, provide insights into the presence and composition of immunologically relevant gene families, including the antigen B- and EG95/45W families, and discuss chemogenomic approaches toward the development of novel chemotherapeutics against cestode diseases. In addition, we discuss the evolution of tapeworm parasites and introduce the research programmes linked to genome initiatives that are aimed at understanding signalling systems involved in basic host-parasite interactions and morphogenesis.

Echinococcus multilocularis as an experimental model in stem cell research and molecular host-parasite interaction.

Totipotent somatic stem cells (neoblasts) are key players in the biology of flatworms and account for their amazing regenerative capability and developmental plasticity. During recent years, considerable progress has been made in elucidating molecular features of neoblasts from free-living flatworms, whereas their role in parasitic species has so far merely been addressed by descriptive studies. Very recently, however, significant advances have been made in the in vitro culture of neoblasts from the cestode Echinococcus multilocularis. The isolated cells proved capable of generating mature metacestode vesicles under laboratory conditions in a manner that closely resembles the oncosphere-metacestode transition during natural infections. Using the established neoblast cultivation protocols, combined with targeted manipulation of Echinococcus genes by RNA-interference, several fundamental questions of host-dependent parasite development can now be addressed. Here, I give an overview of current cultivation techniques for E. multilocularis neoblasts and present experimental approaches to study their function. Furthermore, I introduce the E. multilocularis genome sequencing project that is presently in an advanced stage. The combined input of data from the E. multilocularis sequencing project, stem cell cultivation, and recently initiated attempts to genetically manipulate Echinococcus will provide an ideal platform for hypothesis-driven research into cestode development in the next years.

The management of acute limb ischemia.

Acute limb ischemia (ALI) is one of the most common vascular emergencies and characterized by sudden worsening of limb perfusion mainly caused by embolization of thrombotic masses or acute graft occlusion. It is a serious condition with potential thread to limb viability accompanied by significant mortality, morbidity and costs. This article provides an overview of etiology, classification and treatment options of ALI ischemia with special focus on the issue of postreperfusion syndrome. The concept of reperfusion injury following limb ischemia and a system for controlled limb reperfusion to offset postreperfusion synsrome is described in detail.

Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus.

A fetal goat cell line (ZZ-R 127) supplied by the Collection of Cell Lines in Veterinary Medicine of the Friedrich Loeffler Institute was examined for susceptibility to infection by foot-and-mouth disease (FMD) virus (FMDV) and by two other viruses causing clinically indistinguishable vesicular conditions, namely, the viruses of swine vesicular disease and vesicular stomatitis. Primary bovine thyroid (BTY) cells are generally the most sensitive cell culture system for FMDV detection but are problematic to produce, particularly for laboratories that infrequently perform FMD diagnostic tests and for those in countries where FMD is endemic that face problems in sourcing thyroid glands from FMD-negative calves. Strains representing all seven serotypes of FMDV could be isolated in ZZ-R 127 cells with a sensitivity that was considerably higher than that of established cell lines and within 0.5 log of that for BTY cells. The ZZ-R 127 cell line was found to be a sensitive, rapid, and convenient tool for the isolation of FMDV and a useful alternative to BTY cells for FMD diagnosis.

The influence of host hormones and cytokines on Echinococcus multilocularis signalling and development.

Parasitic helminths display highly complex life-cycles in which the establishment of adults or larvae within host target organs as well as the transition of one developmental stage to the following is influenced by host-derived factors. Due to its approachability concerning in vitro cultivation, the larval stage of the fox-tapeworm Echinococcus multilocularis has recently emerged as a model system to study the molecular nature of such host-derived stimuli and their influence on developmental control in the parasite. Data obtained so far indicate that cytokines which are used by the host for cell-cell communication can also be exploited by the parasite as clues to find suitable target organs. This involves direct interactions of evolutionary conserved signalling systems of the receptor tyrosine--and the receptor serine/threonine-kinase pathways of the parasite with corresponding host cytokines of the insulin-, the epidermal growth factor-, and the transforming growth factor-beta-families. In the present article, we will briefly review in vitro cultivation approaches undertaken so far for E. multilocularis larvae as well as our current knowledge on the parasite's signalling systems and their interaction with host cytokines.

Echinococcus-Host Interactions at Cellular and Molecular Levels.

The potentially lethal zoonotic diseases alveolar and cystic echinococcosis are caused by the metacestode larval stages of the tapeworms Echinococcus multilocularis and Echinococcus granulosus, respectively. In both cases, metacestode growth and proliferation occurs within the inner organs of mammalian hosts, which is associated with complex molecular host-parasite interactions that regulate nutrient uptake by the parasite as well as metacestode persistence and development. Using in vitro cultivation systems for parasite larvae, and informed by recently released, comprehensive genome and transcriptome data for both parasites, these molecular host-parasite interactions have been subject to significant research during recent years. In this review, we discuss progress in this field, with emphasis on parasite development and proliferation. We review host-parasite interaction mechanisms that occur early during an infection, when the invading oncosphere stage undergoes a metamorphosis towards the metacestode, and outline the decisive role of parasite stem cells during this process. We also discuss special features of metacestode morphology, and how this parasite stage takes up nutrients from the host, utilizing newly evolved or expanded gene families. We comprehensively review mechanisms of host-parasite cross-communication via evolutionarily conserved signalling systems and how the parasite signalling systems might be exploited for the development of novel chemotherapeutics. Finally, we point to an urgent need for the development of functional genomic techniques in this parasite, which will be imperative for hypothesis-driven analyses into Echinococcus stem cell biology, developmental mechanisms and immunomodulatory activities, which are all highly relevant for the development of anti-infective measures.

Recent advances in Echinococcus genomics and stem cell research.

Alveolar and cystic echinococcosis, caused by the metacestode larval stages of the tapeworms Echinococcus multilocularis and Echinococcus granulosus, respectively, are life-threatening diseases and very difficult to treat. The introduction of benzimidazole-based chemotherapy, which targets parasite ß-tubulin, has significantly improved the life-span and prognosis of echinococcosis patients. However, benzimidazoles show only parasitostatic activity, are associated with serious adverse side effects and have to be administered for very long time periods, underlining the need for new drugs. Very recently, the nuclear genomes of E. multilocularis and E. granulosus have been characterised, revealing a plethora of data for gaining a deeper understanding of host-parasite interaction, parasite development and parasite evolution. Combined with extensive transcriptome analyses of Echinococcus life cycle stages these investigations also yielded novel clues for targeted drug design. Recent years also witnessed significant advancements in the molecular and cellular characterisation of the Echinococcus 'germinative cell' population, which forms a unique stem cell system that differs from stem cells of other organisms in the expression of several genes associated with the maintenance of pluripotency. As the only parasite cell type capable of undergoing mitosis, the germinative cells are central to all developmental transitions of Echinococcus within the host and to parasite expansion via asexual proliferation. In the present article, we will briefly introduce and discuss recent advances in Echinococcus genomics and stem cell research in the context of drug design and development. Interestingly, it turns out that benzimidazoles seem to have very limited effects on Echinococcus germinative cells, which could explain the high recurrence rates observed after chemotherapeutic treatment of echinococcosis patients. This clearly indicates that future efforts into the development of parasitocidal drugs should also target the parasite's stem cell system.

A gene encoding a superoxide dismutase of the facultative intracellular bacterium Listeria monocytogenes.

A gene (lmsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria.

Transcriptional activation of virulence genes in wild-type strains of Listeria monocytogenes in response to a change in the extracellular medium composition.

A panel of 103 Listeria monocytogenes strains of different origins was examined for haemolysin and lecithinase production in brain-heart infusion (BHI). Three distinct phenotypes were observed. Phenotype 1 was characterized by low to undetectable levels of expression and was exhibited by almost all strains tested. Phenotype 2 expressed high levels of haemolysin and lecithinase and was displayed by five strains: one (P14-A) was a spontaneous mutant derived from a type 1 isolate (P14); the four others (EGD-A, NCTC 7973, SLCC 2373 and CLIP 545) were all laboratory strains kept under in vitro conditions for a long period. Phenotype 3 was intermediate and was exhibited by another laboratory strain (L028). We therefore concluded that phenotype 1 corresponded to the wild type, whereas phenotypes 2 and 3 represented mutant or variant phenotypes. Interestingly, wild-type strains were able to dramatically increase the expression of virulence factors when cultured in BHI treated with activated charcoal (BHIC), up to levels similar to those constitutively expressed by the hyperhaemolytic/lecithinase variants in BHI. Experiments with P14 and P14-A demonstrated that both charcoal and the hyperhaemolytic/lecithinase mutation exerted their effect by inducing (or derepressing) transcription of prfA, the pleiotropic transcriptional activator of the L. monocytogenes virulence regulon. Moreover, P14 and P14-A were equally virulent for mice despite the different levels of virulence factor expression in BHI. Taken together, these observations indicate that L. monocytogenes turns off virulence gene expression when growing in vitro in a rich medium, and suggest that the increased levels of virulence factors in the hyperhaemolytic/lecithinase mutants and in wild-type strains grown in BHIC might represent the levels of expression needed in vivo by L. monocytogenes for infecting host tissues.

The Impella Recover microaxial left ventricular assist device reduces mortality for postcardiotomy failure: a three-center experience.

BACKGROUND: We evaluated patient outcomes and complications associated with the microaxial Impella Recover left ventricular assist device (Impella Cardiosystems AG, Aachen, Germany) for postcardiotomy low-output syndrome. This low-cost device is inserted across the aortic valve through a 10-mm vascular graft sewn to the ascending aorta. METHODS: Impella patients were compared with 198 patients treated with an intraoperative intra-aortic balloon pump between January 2000 and December 2002. Three risk scores were used: the Hausmann score, the Texas Heart Institute score, and the Cleveland intensive care unit score. Between September 2001 and March 2003, 24 patients were treated with the Impella Recover for low-output syndrome. Before device insertion, 21 could not be separated from cardiopulmonary bypass, and 3 had postoperative hemodynamic instability despite high-dose catecholamines. Sixteen were treated with the Impella and intra-aortic balloon pump and 8 with the Impella alone (no intra-aortic balloon pump because of peripheral vascular disease or because deemed unnecessary). RESULTS: No technical problems with device insertion occurred. Pump flow was 3.3 +/- 0.7 L/min at 28,000 +/- 4500 RPM. Support time was 61 +/- 56 hours (range, 7-228 hours). Four devices required repositioning. One device failed (leaking purge line) and was removed. Hemolysis was minimal (lactate dehydrogenase levels of 540 +/- 260 U/dL for Impella survivors). Mortality for Impella patients was 54% (13/24), similar to that for high-risk intra-aortic balloon pump patients (Hausmann score > or =2 [57%], intensive care unit score > or =2 [51%], Texas Heart Institute score > or =0.75 [55%], and cardiac index < or =2.3 [45%]). Cardiac output data were available in 19 Impella patients. Impella patients able to increase their cardiac output to 1 L/min or more above the pump flow of the Impella Recover had a 10% (1/10) mortality, versus 88% (8/9) in patients with a residual cardiac function of 1 L/min or less (P =.001). Comparison of high-risk intra-aortic balloon pump patients with Impella patients with residual cardiac function of 1 L/min or more showed a significant reduction in mortality, regardless of the high-risk definition used. Residual cardiac function was the strongest predictor of survival in Impella patients. CONCLUSIONS: The Impella Recover device provides 3 to 4 L/min flow. It improves survival in patients with low-output syndrome if the heart is able to pump 1 L/min or more above device flow.

The nervous and prenervous roles of serotonin in Echinococcus spp.

Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive and morphogenetic molecule in several metazoan phyla, including flatworms. Serotoninergic nervous system studies are incomplete and 5-HT function/s are unknown in Echinococcus spp., the flatworm parasites that cause hydatid disease. In the present work, we searched for genes of the serotoninergic pathway and performed immunocytochemical and functional analyses of 5-HT in Echinococcus spp. Bioinformatic analysis using the recently available Echinococcus multilocularis and Echinococcus granulosus genomes suggests the presence of genes encoding enzymes, receptors and transporters participating in 5-HT synthesis, sensing and transport in these parasites. However, some components of the pathway could not be identified, suggesting loss or divergence of parasite homologous genes. The serotoninergic neuroanatomy study performed by confocal scanning laser microscopy on different E. granulosus stages showed an increasing level of complexity when the protoscolex develops towards the adult stage and a progressive diminution when the parasite develops towards the metacestode stage. The role of 5-HT as a neurotransmitter in E. granulosus was evaluated by determining the effect of this substance on protoscolex motility. The addition of 5-HT to protoscoleces induced a significant increase in motility for short time periods. Preincubation with 100 µM citalopram, a known 5-HT transporter inhibitor, abolished the 5-HT-induced increase in motility, indicating that the effect could be mediated by a 5-HT transporter. Incubation of protoscoleces with 5-HT for time periods of several days induced a progressive differentiation towards the metacestode stage. The results indicate that 5-HT could have nervous and prenervous roles during Echinococcus spp. development. Taking into account the important roles of 5-HT in parasite biology and the divergence of 5-HT pathway genes with respect to human counterparts, the serotoninergic system could be considered as an amenable drug target against hydatid disease.

High potency vaccines induce protection against heterologous challenge with foot-and-mouth disease virus.

In a series of three homologous and eight heterologous challenge experiments, it was shown that high potency vaccines against foot-and-mouth disease (FMD) serotype A can induce protection even against heterologous challenge infection with viruses that give low r-values with the vaccine strains. The challenge virus specific neutralizing antibody response on the day of challenge (21 days post vaccination) generally correlated with protection.

[The significance of the cation exchange for the cation contents of living Sphagnum]. / Die Bedeutung des Kationenaustausches für den Kationengehalt lebender Sphagnen.

1. A method is given for the determination of the cation exchange capacity (CEC) of small quantities of Sphagnum material (50-100 mg of dry weight, dead material). The method is especially suitable for measurements with bivalent cations. 2. Branches and stems of Sphagnum plants have very different CEC. 3. Under the given conditions the CEC is 11,7 mequiv/100 g of Sphagnum for the monovalent cations Na(+) and K(+) and 23,3 mequiv/100 g of Sphagnum for the bivalent cations Ca(++) and Mg(++). 4. When mono-and bivalent cations are mixed (both 0.01 N), the CEC of the monovalent cation decreases to 1/7-1/40 of the CEC in pure monovalent solution; the CEC of the bivalent cation decreases only very little. 5. The CEC increases with decreasing amounts of Sphagnum in a constant volume of salt-solution; on the other hand it increases with decreasing volumes of salt solution containing a constant amount of Sphagnum. 6. Dead and living material have the same CEC (referred to dry weight). 7. Two methods are given for the separation of the total cation contents of living Sphagnum into the contents of the adherent water, of the cation exchanger and of the living cell. 8. Living Sphagnum contains most of the K(+) and Na(+) within the cells, Mg(++) partly in the cells, partly in the exchanger, and most of the Ca(++) in the exchanger. 9. Living Sphagnum seems to be able to keep the cation contents of the cells rather constant even when the contents of the adherent water and of the exchanger are varying widely.

[Differential diagnosis of granulomatous skin diseases]. / Zur Differentialdiagnose granulomatöser Hautkrankheiten

The case histories of two female patients with granulomatosis disciformis and necrobiosis lipoidica are reported. Widespread skin manifestations, also in face and neck, with peculiar anular lesions complicated the differential diagnostic discrimination from sarcoidosis. The aspects of the cases and their progress are discussed in regard to the necessity of permanent checking of diagnostic preconditions during therapy.