Rapid Rescue Research Articles

NFAT1 in Adult and Cord Blood-Derived Human CD4+ T-Cells: Regulatory Implications for Graft vs. Host Disease (GVHD).

Clinical benefits of umbilical cord blood (UCB) grafts include the lower incidence of acute graft-versus-host disease (aGVHD) following allogeneic transplantation. We have previously demonstrated dramatically lower expression of the Nuclear Factor of Activated T-Cells 1 (NFAT1) protein, its associated transcripts and known binding partners, but not NFAT1 transcripts in UCB CD4+ cells following primary stimulation when compared to T-cells obtained from adult blood (AB). As NFAT1 is a key transcriptional regulator of immune responses, we hypothesize that NFAT1 and NFAT1-dependent factors play a critical role in the increased proliferation and decreased cytokine production seen in UCB, which may contribute to the lower incidence of aGVHD following UCB transplantation observed clinically. We have now demonstrated in vitro via siRNA-mediated knockdown and subsequent analysis of adult-derived primary T-cells with RT-PCR and Cytometric Bead Assay (CBA) that expression of a GVHD-promoting, Th1-type cytokine profile is dependent on expression of NFAT1, and that knockdown of NFAT1 results in a transcriptional and cytokine secretion profile similar to that seen in UCB T-cells with respect to key immunoregulatory cytokines and signal transducers following stimulation. Specifically, siRNA-mediated reduction of NFAT1 by at least 68% results in 93% and 28% reduction of IFN-γ, and TNF-αrespectively, as well as a 55% reduction in CTLA4 measured by RT-PCR. Additionally, short hairpin RNA (shRNA)-mediated stable knockdown of NFAT1 results in an enhanced and prolonged reduction of IFN-γ, IL-2, and TNF-αsecretion over transient siRNA transfection in both the Jurkat cell line and adult primary CD4+ T-cells. However, microarray and RT-PCR time point analyses indicate NFAT1 transcripts are not changed in UCB versus AB CD4+ cells. Under siRNA knockdown conditions, we observed at least a 24-hour delayed decrease in NFAT1 protein levels following mRNA reduction and an eventual rebound of both NFAT1 and its associated cytokines following transient transfection at 48 hours. This suggests a crucial regulatory role for post-translational events which may preferentially occur in UCB-derived T-cells in vivo.Indeed, we have observed ubiquitination of NFAT1 and rapid rescue of NFAT1 expression following treatment with the calpain-proteasome inhibitor Acetyl-L-Leucyl-L-Leucyl-L-Norleucinal (ALLN) in UCB-derived T-cells in vitro by co-immunoprecipitation (co-IP) in a dose-dependent manner. NFAT1 in unstimulated UCB CD4+ cells is shown by co-IP to be significantly more ubiquitinated than in AB-derived cells. Overall, our data indicates that regulation of NFAT1 through ubiquitination and proteasomal degradation, and its subsequent downstream effects may constitute a key difference between T-cells of adult and neonatal origin; specifically that this post-translational modification may serve as a method by which UCB-derived T-cells may prevent NFAT1 activity, impacting their allogeneic response.

Generation of VP5 deficient mutant of infectious bursal disease virus strain HZ2

Infectious bursal disease virus (IBDV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The nonstructural protein VP5 has been reported to be associated with virus-induced cell apoptosis and pathogenicity, but its role in viral replication has not been unequivocally identified. Based on a PCR introduced mutagenesis strategy, the 33 bp of 96–129 bp located between ORF A1 and ORF A2 of genomic segment A of IBDV strain HZ2 were deleted, and an Nhe I (GcTaGc) site was inserted at 96–102 bp simultaneously. The mutated segment A was ligated into pCI, resulting in pCI-ANhe3. A chimeric and deficient IBDV strain, named strain ANhe3, was recovered from chicken embryo fibroblast (CEF) cells by co-transfection with pCI-ANhe3 and pCI-mB, derived from the genomic segment B strain HZ2. The indirect fluorescent assay identified that strain ANhe3 could replicate on CEF cells without expression of VP5. Further examination showed that the pathogenesis of strain ANhe3 replicating on SPF chicken embryos was attenuated compared to strain HZ2. This paper provides a new rapid rescue strategy for gene-deleted virus. This strategy lays a basis for gene-deleted vaccine of IBDV.

MAPK Signaling 2: Reversible Rescue

Chemical rescue of catalytically defective mutant enzymes has been a productive approach to studying enzyme function in vitro, but applications of the technique in vivo have so far met with limited success. Qiao et al. have achieved rapid and reversible rescue of the protein tyrosine kinase Src in live cells using the small molecule imidazole. The work provides insight into the mitogen-activated protein (MAP) kinase signaling pathway, including identifying several new Src substrates. Besides being a useful tool for studying cell signaling, small molecules that rescue disease-related mutant enzymes may have therapeutic potential. Y. Qiao, H. Molina, A. Pandey, J. Zhang, P. A. Cole, Chemical rescue of a mutant enzyme in living cells. Science 311 , 1293-1297 (2006). [Abstract] [Full Text]

Functional study of the Nha1p C-terminus: involvement in cell response to changes in external osmolarity

Saccharomyces cerevisiae uses different mechanisms to adapt to changes in environmental osmolarity. Upon hyperosmotic shock, cells first mobilize a rapid rescue system that prevents excessive loss of ions and water; then in the adaptation period they accumulate a compatible solute (glycerol). When subjected to hypoosmotic shock, they rapidly release intracellular stocks of glycerol to reduce intracellular osmolarity and prevent bursting. The plasma membrane Nha1 alkali metal cation/H+ antiporter is not important in helping the cells to survive a sudden drop in external osmolarity, but is involved in the cell response to hyperosmotic shock. For this role, its long hydrophilic C-terminus is indispensable. The capacity of the Nha1 antiporter to transport potassium is regulated by Hog1 kinase. Upon sorbitol-mediated stress, the Nha1p potassium export activity decreases in order to maintain a higher intracellular concentration of solutes. The C-terminal-less Nha1 version is not inactivated and its potassium efflux activity renders cells very sensitive to hyperosmotic shock. Taken together, our results suggest an important role of Nha1p and its C-terminus in the immediate response to hyperosmotic shock as part of the rapid rescue mechanism.

A comparison of the effects of oral montelukast and inhaled salmeterol on response to rescue bronchodilation after challenge

Objectives: To compare the effects of addition of montelukast or salmeterol to inhaled corticosteroids (ICS) on the response to rescue beta2-agonist use after exercise-induced bronchoconstriction. Methods: A double-blind, placebo-controlled study was performed at 16 centers in the United States. Patients with asthma ( n=122, ages 15–58) whose symptoms were uncontrolled on low-dose inhaled fluticasone and who had a history of exercise-induced worsening of asthma were randomized to receive either montelukast (10 mg once daily), salmeterol (50 μg twice daily), or placebo for 4 weeks. Standardized spirometry after exercise challenge and beta2-agonist rescue was performed at baseline, week 1 and 4. Results: Maximum achievable forced expiratory volume in 1 s (FEV 1) percent predicted after rescue beta2-agonist improved in the montelukast (+1.5%) and placebo (+1.2%) groups at 4 weeks, but diminished in the salmeterol (−3.9%) group ( P<0.001). Although pre-exercise FEV 1 was greatest with salmeterol ( P=0.10), patients taking montelukast had significantly greater protection from an exercise-induced decrease in FEV 1 than those taking salmeterol ( P<0.001). Both the magnitude and rate of rescue bronchodilation were greater with montelukast compared with salmeterol ( P<0.001). Five minutes after rescue beta2-agonist, 92% of patients taking montelukast and 68% of those taking placebo had recovered to pre-exercise levels, whereas only 50% of those taking salmeterol had recovered to pre-exercise levels. Conclusion: In patients whose asthma symptoms remain uncontrolled using ICS, addition of montelukast permits a greater and more rapid rescue bronchodilation with a short-acting beta2-agonist than addition of salmeterol and provides consistent and clinically meaningful protection against exercise-induced bronchoconstriction.

Medical considerations in the use of helicopters in mountain rescue.

The outcome of patient care can be dramatically improved by bringing rapid rescue and medical care to the mountain rescue scene and by rapid transport to a medical facility. The use of a helicopter for these purposes is common. It is necessary when it has clear advantages for victims in comparison with ground rescue and transport. Helicopters should work within the existing emergency medical system and must be staffed by appropriate mountain rescue and medically trained personnel. Activation time should be as short as possible. Activation of a helicopter for a mountain rescue should primarily include indication and assessment of flight and safety conditions. No other mediators or delaying factors should be permitted. The main safety criteria are appropriate mountain rescue and flight training, competence of air and ground crews, radio communication between the air and ground crews, and mission briefing before the rescue. Criteria for a helicopter used for mountain rescue are proper medical and rescue equipment, load capacity, adequate space, and others. There are two main groups of indications for use of a helicopter for mountain rescue: the patient's condition and the circumstances at the site of the accident. All persons responsible for the activation of the helicopter rescue operation should be aware of specific problems in the mountains or wilderness.

Corpus luteum response to exogenous HCG during the mid-luteal phase of the menstrual cycle.

To assess a range of exogenous HCG regimes designed to simulate the endocrine environment occurring in biochemical, single and multiple pregnancies and to study the response of the corpus luteum to those regimes. Prospective clinical study. Twenty-five normally cycling women aged 24-35 years were given one of four regimes of HCG injections designed to mimic the HCG concentrations found following spontaneous implantation. Regimes A, B, C and D were designed with starting HCG doses of 60, 140, 250 and 1000 iu, respectively. The daily HCG injections were then increased to give a doubling concentration every 30 h for regime A, every 27 h for regime B, every 24 h for regimes C and D. HCG administration was started on either days 7 or 8 after the LH peak. Plasma HCG and progesterone concentrations. Subjects given regime A failed to demonstrate any rescue of the corpus luteum despite low-detectable amounts of HCG in the circulation equivalent to those seen in some biochemical pregnancies. In contrast, subjects given regimes B and C demonstrated prompt increases in progesterone secretion immediately after the first HCG injection achieving HCG and progesterone concentrations in plasma similar to those seen in normal singleton pregnancies. Subjects given regime D also showed rapid rescue of the corpus luteum but this time achieved plasma HCG concentrations in the range normally seen in multiple pregnancies. All subjects in regimes B, C and D secreted significantly higher amounts of progesterone than those in regime A (P<0.001). However, despite the greater amounts of HCG used in regime D, the amount of progesterone produced was not significantly different from regimes B or C. The exogenous HCG regimes used in this study successfully mimicked the hormonal environment found in biochemical, single and multiple pregnancies and elicited appropriate corpus luteum responses.

Efficient recovery and regeneration of integrated retroviruses.

We report a rapid and efficient PCR-based rescue procedure for integrated recombinant retroviruses. Full-length proviral DNA is amplified by long-range PCR using a pair of primers derived from the long terminal repeats (LTR), and virus is regenerated by transfecting retrovirus-packaging cells with the PCR-derived provirus. The viral yield from the PCR product is similar to that from the retroviral plasmid vector, and the representation of different inserts is accurately maintained in the recovered retroviral population. This procedure is suitable for expression cloning from retroviral libraries and should be applicable to the analysis of natural retrovirus populations.

The Russian-Japanese "Pazyryk" Programme the First Year of Joint Studies

AbstractThe early Iron Age tumuli of the Altai mountains are unique in preserving largely intact frozen corpses, horses, textiles, leather and wood of the Scythian period. Further such burials exist: in 1991 a reconnaissance excavation of a frozen tumulus at Ak-Alakh again yielded a fabulous inventory. However the conditions of their preservation require their rapid rescue and conservation and adequate funding to do so. In 1991 the USSR and Japan signed an agreement for the joint study of the tumuli of the Pazyryk culture, which included provisions for the conduct of field research on the Ukok plateau, the restoration and conservation of materials from the tumuli (Japan has made Yens 100 m available for this purpose), and the transport of the excavated tumuli to musea. A large tumulus of the 5th-3rd c. B.C. at Kurtuguntas has since been excavated, the unique material recovered was conserved on site and the burial construction transported to the museum at Akademgorodok. In the Bertok valley 60 sites were surveyed and 15 excavated (only one tulumus had been disturbed in antiquity). A monograph is in preparation.

Rapid Rescue of Cellular Transcriptional Activator Elements by Amplification of a Single Copy Selection Gene

Cellular transcriptional activator sequences from a Syrian hamster cell line (baby hamster kidney (BHK] were rescued by a double selection procedure. An enhancer-deficient SV40 promoter was linked to the neomycin resistance (NEO) gene and transfected into BHK cells. Genomic DNA fragments of G418-resistant cell clones containing multiple copies of integrated plasmid DNAs were used for a second transfection of BHK cells, resulting in the genomic integration of a single copy plasmid which expresses the NEO gene efficiently. For rapid cloning of the integrated promoter and adjacent cellular DNA sequences, these cell clones were fused to COS-1 cells, thereby providing SV40 large T antigen and the monkey cell permissive factor necessary for SV40 replication. Resulting from this fusion, the integrated plasmid and adjacent sequences were amplified to about 1000 extrachromosomal copies giving rise to an abundant pool of promoter elements which thus can be cloned into a plasmid very easily for further investigations. Promoter analyses of three clones in the chloramphenicol acetyltransferase transient expression assay demonstrated that the recombination with cellular DNA enables the initially defective SV40 promoter to express at wild type levels.

Helicopter Extractable Cold Weather Life Raft—A Design and Development History

In military assaults on hostile shores made jointly by troop-carrying helicopters and landing craft, some vehicles will be damaged by enemy fire, forcing their surviving personnel into the sea. These combat survivors, suddenly exposed to cold water, will in minutes be subject to rapid muscular immobilization followed by immediate death from immersion hypothermia. Conventional methods and equipment for search and rescue are inadequate in providing a rapidly deployed, thermally protected, flotation platform effective enough to keep multiple numbers of survivors alive until rescued. This inability to rapidly rescue multiple numbers of disaster victims is present on land as well. High-rise building fires and inaccessible areas such as mountains pose a challenge to rapid rescue methods utilizing aircraft. The authors of this paper examine the present methods and equipment utilized in aircraft search and rescue. By creating scenarios of various possible events, methods are suggested to improve techniques by using a helicopter-extractable cold weather life raft (HECWLR). The HECWLR was designed, prototypes constructed, and successfully tested. The HECWLR potentially provides the means of rapidly rescuing disaster survivors in multiple numbers on both sea and on land.

Rapid rescue of the defective M-MSV genome by the use of cell fusion

Rapid rescue of the defective M-MSV genome by the use of cell fusion

Terracotta Figures from Entebbe, Uganda

In I964 two broken terracotta figures were found during excavations for the foundations of a new garage at the Geological Survey in Entebbe, Uganda. Except for a broken figure from Kanyanya hill, described below, these are the first terracotta figures to be found in east Africa since the Luzira head described by Wayland et al. (1933). Though a rapid rescue excavation' was undertaken, no further figures were found and the exact context of the figures is unknown. As the figures are unique in east Africa it is hoped that this brief report will enable parallels to be located outside Uganda.