Fungal Spores Viability on the International Space Station.

In this study we investigated the security of a spaceflight experiment from two points of view: spreading of dried fungal spores placed on the different wafers and their viability during short and long term missions on the International Space Station (ISS). Microscopic characteristics of spores from dried spores samples were investigated, as well as the morphology of the colonies obtained from spores that survived during mission. The selected fungal species were: Aspergillus niger, Cladosporium herbarum, Ulocladium chartarum, and Basipetospora halophila. They have been chosen mainly based on their involvement in the biodeterioration of different substrate in the ISS as well as their presence as possible contaminants of the ISS. From biological point of view, three of the selected species are black fungi, with high melanin content and therefore highly resistant to space radiation. The visual inspection and analysis of the images taken before and after the short and the long term experiments have shown that all biocontainers were returned to Earth without damages. Microscope images of the lids of the culture plates revealed that the spores of all species were actually not detached from the surface of the wafers and did not contaminate the lids. From the adhesion point of view all types of wafers can be used in space experiments, with a special comment on the viability in the particular case of iron wafers when used for spores that belong to B. halophila (halophilic strain). This is encouraging in performing experiments with fungi without risking contamination. The spore viability was lower in the experiment for long time to ISS conditions than that of the short experiment. From the observations, it is suggested that the environment of the enclosed biocontainer, as well as the species'specific behaviour have an important effect, reducing the viability in time. Even the spores were not detached from the surface of the wafers, it was observed that spores used in the long term experiment lost the outer layer of their coat without affecting the viability since they were still protected by the middle and the inner layer of the coating. This research highlights a new protocol to perform spaceflight experiments inside the ISS with fungal spores in microgravity conditions, under the additional effect of possible cosmic radiation. According to this protocol the results are expressed in terms of viability, microscopic and morphological changes.

Effective treatment of patients in a deep salvage situation with "non-active HAART": experiences with the expert advice system RADATA.

BACKGROUND: Clinical studies suggest expert recommendations as a possibility to optimize highly active antiretroviral therapy (HAART) in patients with multi-drug resistant virus strains. An online system (RADATA) has been developed to provide expert advice for the drug therapy of HIV-infected patients. OBJECTIVE: To evaluate the efficacy of expert-advice-guided HAART switches in patients with triple-class failure. METHODS: Virological and immunological outcome of patients having undergone at least three prior ART regimens, including nucleoside inhibitor (NRTI), nonnucleoside reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) use, were analyzed. Changes in HIV-RNA and CD4-cell count were evaluated every 3 months. RESULTS: 183 patients with a median baseline viral load of 3.90 log copies/ml (1.88-6.54 log) and a CD4-cell count of 298 c/ll (5-910 c/ll) were eligible for analysis. The patients had a median of seven prior ART regimens and a treatment duration of 83 months. A median of three (range 0-8) NRTI-, two (0-7) thymidine-associated (TA), one (0-4) NNRTI-, and three (0-13) PI-associated resistance mutations were present at baseline. Despite available resistance analyses and expert recommendations, 66% (n = 119) of the patients started a new ART regimen without any active drugs according to the resistance analysis. The HIV-RNA declined by a median of 0.61 log and 0.92 log after 12 and 24 months, respectively, while the CD4-cell count rose by a median of +9 c/microl and +25 c/microl during this period. No significant differences related to number of prior regimens or number of active substances used could be found. CONCLUSION: Despite extensive pre-treatment and multiple resistances against prescribed HAART, our patients demonstrated a decline in viral load and a stable CD4-cell count over the observation period. We conclude that the activity of antiretroviral regimens is not exclusively explained by the current algorithms used for estimating antiretroviral drug activity.

Porcine circovirus 2 inclusion bodies in pulmonary and renal epithelial cells.

Porcine circovirus 2 (PCV2) is the cause of postweaning multisystemic wasting syndrome (PMWS). The most common lesions of PMWS are lymphohistiocytic to granulomatous lymphadenitis, interstitial pneumonia and interstitial nephritis, with intracytoplasmic amphophilic botryoid inclusion bodies in macrophages. In addition to these typical changes, intracytoplasmic botryoid inclusion bodies were observed in bronchial, bronchial glandular, and renal tubular epithelium of several pigs from 4 different farms in Western and Eastern Canada. PCV2 inclusion bodies were demonstrated to be located in the cytoplasm of epithelial cells by immunohistochemical staining for PCV2 and cytokeratin antigens and by ultrastructural demonstration of viral particles in the inclusion bodies within renal tubular epithelium.

[Multiple diabetic muscular infarctions after liver and kidney transplantation: a case report]. / Multiples infarctus musculaires diabétiques après transplantation hépatique et rénale.

INTRODUCTION: Several neuromuscular diseases may complicate diabetes mellitus and transplantation, including chronic sensorimotor length dependent polyneuropathy. OBJECTIVE: Description of muscular infarction, a rare complication of diabetes mellitus, which occurred after liver and kidney transplantation. CASE REPORT: A 57-year-old patient presented with long-term diabetes mellitus and multiple complications. End-stage renal and hepatic disease led to kidney and liver transplantation. Twenty-seven days after transplantation, swelling and induration appeared in the left shoulder and forearm. Forty-three days after transplantation, the same symptoms appeared in both lower limbs. Markedly reduced range of motion led to severe disability. Bone scans showed multiple spots following muscle anatomy. Computed tomography gave negative results. Magnetic resonance imaging (MRI) confirmed muscular infarction by a high T1 signal (muscular necrosis) and soft-tissue infiltration. DISCUSSION AND CONCLUSION: Muscular infarction is a rare and unknown complication of diabetes mellitus. It is characterised by sudden painful muscular induration and swelling affecting one muscle at a time with recurrence. Our patient presented with simultaneous multiple muscular infarctions in 3 limbs. Diagnosis was based on clinical investigation and MRI. The treatment is conservative and the condition generally resolved by itself. However, the long-term prognosis of muscular infarction is not good because of the cardiovascular-associated complications of diabetes mellitus.

Space bioreactors: their use, their future.

NASA: The use of bioreactors during space flight is discussed. The major elements of a bioreactor are a culture chamber, sensors, a control unit with feedback, as gas exchange system, a pump, fresh culture medium, and a waste reservoir. Types of bioreactors developed for use in space include the Woodlawn Wanderer 9 apparatus, the Space tissue loss system, rotating wall vessel, dynamic cell culture system and the SBR I. Future development for space bioreactors include improvements for cultivation of mammalian cells and tissue engineering and the transfer of bioreactor technology for earth-bound instruments.^ieng

Performance of a miniaturized bioreactor in space flight: microtechnology at the service of space biology.

We describe here the performance and the use of microtechnology in a miniaturized bioreactor developed for the continuous cultivation of yeast cells, Saccharomyces cerevisiae, in microgravity. This bioreactor has been used on two Shuttle missions, where its functionality was successfully demonstrated. In the future, bioreactors will become a key element for long-term experiments, and would also be applied in the cultivation of mammalian cells or tissues for medical applications.

Essential role of Gab1 for signaling by the c-Met receptor in vivo.

The docking protein Gab1 binds phosphorylated c-Met receptor tyrosine kinase directly and mediates signals of c-Met in cell culture. Gab1 is phosphorylated by c-Met and by other receptor and nonreceptor tyrosine kinases. Here, we report the functional analysis of Gab1 by targeted mutagenesis in the mouse, and compare the phenotypes of the Gab1 and c-Met mutations. Gab1 is essential for several steps in development: migration of myogenic precursor cells into the limb anlage is impaired in Gab1-/- embryos. As a consequence, extensor muscle groups of the forelimbs are virtually absent, and the flexor muscles reach less far. Fewer hindlimb muscles exist, which are smaller and disorganized. Muscles in the diaphragm, which also originate from migratory precursors, are missing. Moreover, Gab1-/- embryos die in a broad time window between E13.5 and E18.5, and display reduced liver size and placental defects. The labyrinth layer, but not the spongiotrophoblast layer, of the placenta is severely reduced, resulting in impaired communication between maternal and fetal circulation. Thus, extensive similarities between the phenotypes of c-Met and HGF/SF mutant mice exist, and the muscle migration phenotype is even more pronounced in Gab1-/-:c-Met+/- embryos. This is genetic evidence that Gab1 is essential for c-Met signaling in vivo. Analogy exists to signal transmission by insulin receptors, which require IRS1 and IRS2 as specific docking proteins.

Human immune cells as space travelers.

Experiments in space have shown that T lymphocyte function is altered in more than 50% of space crew members. There is strong evidence that such effect is due to stress rather than to weightlessness per se. However the health of astronauts was never threatened so far. Experiments in-vitro with cultures of human peripheral blood lymphocytes (not from astronauts) have shown that T cell function is dramatically reduced. Recent work with the random positioning machine, a new instrument to simulate conditions similar to microgravity, indicate that there are direct gravitational effects on the genetic expression of interleukin-2 and of its receptor in T lymphocytes.

Microtechnology in space bioreactors.

Space biology is a young and rapidly developing discipline comprising basic research and biotechnology. In the next decades it will play a prominent role in the International Space Station (ISS). Therefore, there is an increasing demand for sophisticated instrumentation to satisfy the requirements of the future projects in space biology. Bioreactors will be needed to supply fresh living material (cells and tissues) either to study still obscure basic biological mechanisms or to develop profitable bioprocesses which will take advantage of the peculiar microgravity conditions. Since more than twenty years, the Space Biology Group of the ETHZ is carrying out research projects in space (Space Shuttle/Spacelab, MIR Station, satellites, and sounding rockets) that involve also the development of space-qualified instrumentation. In the last ten years we have developed, in collaboration with Mecanex SA, Nyon, and the Institute of Microtechnology of the University of Neuchatel, a space bioreactor for the continuous culture of yeast cells under controlled conditions. Sensors, pH control, nutrients pump and fluid flowmeter are based on state-of-the-art silicon technology. After two successful space flights, a further improved version is presently prepared for a flight in the year 2000.

Simulated microgravity inhibits the genetic expression of interleukin-2 and its receptor in mitogen-activated T lymphocytes.

Experiments conducted in space in the last two decades have shown that T lymphocyte activation in vitro is remarkably reduced in microgravity. The data indicate that a failure of the expression of the interleukin-2 receptor (measured as protein secreted in the supernatant) is responsible of the loss of activity. To test such hypothesis we have studied the genetic expression of interleukin-2 and of its receptor in concanavalin A-activated lymphocytes with the RT-PCR technology. Microgravity conditions were simulated in the fast rotating clinostat and in the random positioning machine. The latter is an instrument introduced recently to study gravitational effects on single cells. Our data clearly show that the expression of both IL-2 and IL-2Ralpha genes is significantly inhibited in simulated O X g. Thus full activation is prevented.

Cultivation of Saccharomyces cerevisiae in a bioreactor in microgravity.

Yeast cells were cultured for 8 d in a newly developed bioreactor during the Spacelab IML-2 mission. Two bioreactors, one stirred and one without stirring, were installed in the Biorack facility in space. Two control units were installed in the Biorack module at the Kennedy Space Center. Samples were drawn on mission day 3, 5, 6, 7 and 8 and preserved either by freezing or chemically fixed for post-flight analysis. The values of pH, pH regulation, temperature and redox potential were transmitted on-line to the ground station throughout the mission. The performance of the bioreactor was satisfactory except for a partial failure of the medium micropump. Despite the failure of the pump, the data support the following conclusions: There is a significant difference in the distribution of the bud scars between cells cultured at 0 x g and at 1 x g. The percentage of randomly distributed bud scars was significantly higher in the flight (17%) than in the ground control cells (5%). No remarkable differences were noted in the cell cycle, ultrastructure, cell proliferation, cell volume, ethanol production and glucose consumption.

Motogenic and morphogenic activity of epithelial receptor tyrosine kinases.

Receptor tyrosine kinases play essential roles in morphogenesis and differentiation of epithelia. Here we examined various tyrosine kinase receptors, which are preferentially expressed in epithelia (c-met, c-ros, c-neu, and the keratin growth factor [KGF] receptor), for their capacity to induce cell motility and branching morphogenesis of epithelial cells. We exchanged the ligand-binding domain of these receptors by the ectodomain of trkA and could thus control signaling by the new ligand, NGF. We demonstrate here that the tyrosine kinases of c-met, c-ros, c-neu, the KGF receptor, and trkA, but not the insulin receptor, induced scattering and increased motility of kidney epithelial cells in tissue culture. Mutational analysis suggests that SHC binding is essential for scattering and increased cell motility induced by trkA. The induction of motility in epithelial cells is thus an important feature of various receptor tyrosine kinases, which in vivo play a role in embryogenesis and metastasis. In contrast, only the c-met receptor promoted branching morphogenesis of kidney epithelial cells in three-dimensional matrices, which resemble the formation of tubular epithelia in development. Interestingly, the ability of c-met to induce morphogenesis could be transferred to trkA, when in a novel receptor hybrid COOH-terminal sequences of c-met (including Y14 to Y16) were fused to the trkA kinase domain. These data demonstrate that tubulogenesis of epithelia is a restricted activity of tyrosine kinases, as yet only demonstrated for the c-met receptor. We predict the existence of specific substrates that mediate this morphogenesis signal.

Development of a miniature bioreactor for continuous culture in a space laboratory.

A new type of miniature bioreactor for continuous culture of yeast cells in space laboratories has been developed. Silicon microtechnology has permitted the integration of numerous functions and systems in a volume of 87 x 63 x 63 mm3 and a weight of 610 g. The 100 ml of fresh medium can be delivered at variable flow rates to the cultivation chamber (volume 3 ml) by means of a micropump. The culture is agitated by a magnetic stirrer. Microsensors monitor pH, temperature and redox potential. The decrease of pH occurring during the cultivation of Saccharomyces cerevisiae is compensated electrochemically. A window allows the inspection of the culture status. Samples of up to 1 ml can be drawn through a silicone rubber septum. The data measured by the sensors are transmitted on-line to the ground station during operations in space. The bioreactor had to fulfil several requirements related to the safety regulation of the space agencies. In particular, new materials had to be selected and tested for their biocompatibility. The instrument has now passed all space and biological qualification tests and will be used in an experiment selected by ESA for the International Microgravity Laboratory-2 Mission in Spacelab in July 1994. This paper gives the results of the functional and biological tests and a detailed description of the instrument.

Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium.

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.

Characterization of lignin peroxidase-encoding genes from lignin-degrading basidiomycetes.

Two closely linked lignin peroxidase (LPO)-encoding genes (lpo) from Phanerochaete chrysosporium were isolated. Nucleotide sequence studies indicated that the two genes are separated by 1.3 kb of flanking DNA and transcribed in opposite directions. Cloned P. chrysosporium lpo gene probes have been shown to hybridize to multiple sequences present in the DNAs of the white-rot fungi, Bjerkandera adusta, Coriolus versicolor and Fomes lignosus, but no hybridization was detected with DNA from Pleurotus ostreatus. Thus, lpo gene families appear to be common in a number of lignin-degrading basidiomycetes, some of which have not yet been shown to produce LPO proteins.

Direct comparison of electric field-mediated and PEG-mediated cell fusion for the generation of antibody producing hybridomas.

As an alternative to polyethylene glycol (PEG), electric field pulses offer, in theory, fusion conditions whose parameters are better controllable. In 1985 (1) we reported on the successful generation of hybridoma clones by means of electrofusion performed in a batch-type manner similar to that usually employed with PEG, and applicable to any type of antigens. Here we summarize the results of a series of fusions performed since then in which both electric field and PEG induced fusion were directly compared. Different types of antigens were used. Electrofusion resulted in a 3.8 to 33.0 times higher yield of hybridomas per unit number of spleen cells. Moreover, hybridomas grew more vigorously after fusion and, therefore, were earlier visible. Other parameters examined revealed no differences between hybridomas generated by either method.

Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene.

The basidiomycete fungus Phanerochaete chrysosporium produces a number of extracellular peroxidases which appear to be important for lignin degradation. We present here the isolation and complete nucleotide (nt) sequence of a gene (lpo) coding for lignin peroxidase (LPO), the coding region of which is identical to a lpo cDNA sequence which had previously been described [M. Tien and C.-P.D. Tu, Nature 326 (1987) 520-523]. The deduced amino acid (aa) sequence corresponds to 372 aa residues and the coding region is interrupted by eight short introns that range in size from 50 to 62 nt. Southern blot experiments using the cloned lpo gene as hybridization probe revealed a complex restriction fragment pattern, indicating that there are a number of lpo-related nucleotide sequences present in P. chrysosporium DNA which cross-hybridize. We also present data on the in vivo expression of the lpo genes and show that they are regulated at the RNA level and that the structure of the transcripts as judged from S1 experiments is complex. These data are consistent with the idea that there are a number of related lpo genes in P. chrysosporium which constitute a gene family.

Cross-neutralizing antibodies induced by single serotype vaccination of cows with rotavirus.

Single serotype vaccination of mature cows with nine different strains of bovine, simian and human rotaviruses induced heterotypic milk and serum neutralizing antibodies against two bovine and four human rotavirus serotypes. Immunization with single-shelled simian rotavirus SA11 increased milk and serum neutralization titres fivefold over those of control cows, without inducing antibodies to outer shell polypeptides of rotavirus. Vaccination with double-shelled SA11 virions also elicited cross-reacting antibodies to the outer shell proteins VP3 and/or VP7 which neutralized rotavirus seven times more efficiently than antisera to single-shelled SA11 virus. A related rotavirus similar to simian rotavirus SA11, but from a different host, might thus be an attractive vaccine for immunization of pregnant cows to confer passive immunity to calves.

Bovine milk immunoglobulins for passive immunity to infantile rotavirus gastroenteritis.

Pregnant cows were successfully hyperimmunized with all four human rotavirus serotypes, resulting in a 100-fold increase in neutralizing milk antibody titers over those of controls. Milk antibodies were isolated batchwise from 1,000 kg of pooled milk for the first 10 lactation days, yielding 10 kg of freeze-dried milk immunoglobulin concentrate consisting of 50% bovine milk immunoglobulins. Milk immunoglobulin concentrate showed neutralizing activities against all four human rotavirus serotypes that were 100 times higher than those in pooled human milk samples and 10 times higher than those in a commercial pooled immunoglobulin preparation from pooled human blood serum. In vitro neutralization tests showed that milk immunoglobulin concentrate had powerful antiviral activity, even against very high doses of infectious rotaviruses. Because the technology of the milk immunoglobulin concentrate ensures that it is innocuous and can be used for oral application, it is proposed that milk immunoglobulin concentrate be used to induce passive immunity to infantile rotavirus gastroenteritis.