Demonstration of a CW diode-pumped Ar metastable laser operating at 4 W.

Optically pumped rare gas lasers are being investigated as potential high-energy, high beam quality systems. The lasing medium consists of rare gas atoms (Rg=Ne, Ar, Kr, or Xe) that have been electric discharge excited to the metastable np5(n+1)s P32 state. Following optical excitation, helium (He) at pressures of 200-1000 Torr is used as the energy transfer agent to create a population inversion. The primary technical difficulty for this scheme is the discharge production of sufficient Rg* metastables in the presence of >200 Torr of He. In this Letter, we describe a pulsed discharge that yields >1013 cm-3Ar* in the presence of He at total pressures up to 750 Torr. Using this discharge, a diode-pumped Ar* laser providing 4.1 W has been demonstrated.

Dose-specific efficacy of Haemophilus influenzae type b conjugate vaccines: a systematic review and meta-analysis of controlled clinical trials.

Global coverage of infant Haemophilus influenzae type b (Hib) vaccination has increased considerably during the past decade, partly due to GAVI Alliance donations of the vaccine to low-income countries. In settings where large numbers of children receive only one or two vaccine doses rather than the recommended three doses, dose-specific efficacy estimates are needed to predict impact. The objective of this meta-analysis is to determine Hib vaccine efficacy against different clinical outcomes after receiving one, two or three doses of vaccine. Studies were eligible for inclusion if a prospective, controlled design had been used to evaluate commercially available Hib conjugate vaccines. Eight studies were included. Pooled vaccine efficacies against invasive Hib disease after one, two or three doses of vaccine were 59%, 92% and 93%, respectively. The meta-analysis provides robust estimates for use in decision-analytical models designed to predict the impact of Hib vaccine.

Density functional theory (DFT) study of edaravone derivatives as antioxidants.

Quantum chemical calculations at the B3LYP/6-31G* level of theory were employed for the structure-activity relationship and prediction of the antioxidant activity of edaravone and structurally related derivatives using energy (E), ionization potential (IP), bond dissociation energy (BDE), and stabilization energies (ΔE(iso)). Spin density calculations were also performed for the proposed antioxidant activity mechanism. The electron abstraction is related to electron-donating groups (EDG) at position 3, decreasing the IP when compared to substitution at position 4. The hydrogen abstraction is related to electron-withdrawing groups (EDG) at position 4, decreasing the BDE(CH) when compared to other substitutions, resulting in a better antioxidant activity. The unpaired electron formed by the hydrogen abstraction from the C-H group of the pyrazole ring is localized at 2, 4, and 6 positions. The highest scavenging activity prediction is related to the lowest contribution at the carbon atom. The likely mechanism is related to hydrogen transfer. It was found that antioxidant activity depends on the presence of EDG at the C(2) and C(4) positions and there is a correlation between IP and BDE. Our results identified three different classes of new derivatives more potent than edaravone.

Identification of a lymphokine that stimulates eosinophil differentiation in vitro. Its relationship to interleukin 3, and functional properties of eosinophils produced in cultures.

Factors stimulating eosinophil differentiation in vitro have been studied by means of a liquid bone marrow culture system in which the number of eosinophils is estimated directly by morphology or indirectly by assay for eosinophil peroxidase. The results show that eosinophil colonies are not formed in agar, emphasizing the importance of the liquid culture system. Three types of evidence identify a novel lymphokine, eosinophil-differentiating factor (EDF). (a) Coordinate analysis of lymphokine activity in media conditioned by a panel of parasite antigen and another panel of alloantigen-reactive T cell clones indicates that EDF is distinct from interleukin 2 (IL-2), IL-3, and bone marrow proliferation activity (BMPA). (b) A T hybrid (NIMP-TH1) produces EDF but no IL-2, IL-3, interferon, or colony-stimulating factor. (c) Gel filtration of conditioned media (CM) indicates that NIMP-TH1 and a T clone (NIMP-T2) produce EDF (Mr 46,000). NIMP-T2 also produced IL-3 (Mr 26,000) but this was easily separated from EDF. IL-3 is also shown to have eosinophil differentiation activity (EDA) but this represents a very small proportion of the EDA in T2-CM. Fractionation of WEHI-3-CM indicates that EDA from this source has a similar elution profile to IL-3 (Mr 35-36,000). Furthermore, a comparison of the relative activities in purified IL-3 and WEHI-3-CM indicates that all the EDA can be attributed to the IL-3 in the latter. EDF is shown to stimulate production of eosinophils in long-term bone marrow cultures; the kinetics of eosinophil production suggests that EDF is acting on committed precursors in the bone marrow. The transient nature of eosinophil production suggests that precursors from multipotential stem cells are not produced. The eosinophils produced in these cultures are morphologically normal and functional in that they lysed sheep red blood cells coated with IgG1, IgG2a, and IgG2b, but not with IgM, IgA, or IgE. In addition, they were capable of adhering to and killing Schistosoma mansoni schistosomula.

Eosinophilia in transgenic mice expressing interleukin 5.

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

Murine eosinophil differentiation factor. An eosinophil-specific colony-stimulating factor with activity for human cells.

A purified murine lymphokine, eosinophil differentiation factor (EDF), was found to be a selective stimulus for the clonal proliferation and differentiation of murine eosinophil progenitor cells, establishing it as the murine eosinophil colony-stimulating factor (Eo-CSF). EDF was also active on human eosinophil progenitors and mature blood eosinophils, but had no effect on neutrophil or macrophage precursor cells, nor on blood neutrophils. In culture of human bone marrow cells, EDF stimulated equal numbers and equal sizes of eosinophil colonies to develop when compared with human placental conditioned medium, a source of human CSFs, suggesting that all responsive progenitor cells were stimulated. Clone transfer experiments and the linear relationship between number of bone marrow cells plated and colonies produced confirmed that the action of EDF was directly on eosinophil progenitor cells. EDF increased the capacity of human blood eosinophils, but not neutrophils, to kill antibody-coated tumor cells and to phagocytose serum-opsonized yeast cells. This functional activation was associated with the enhanced expression of functional antigens (GFA-1, GFA-2, and the receptor for C3bi) on eosinophils. The possession by EDF (Eo-CSF) of all the properties expected of a human eosinophil CSF raises the possibility that a human analog of this molecule exists, and is involved in the regulation of production and function of human eosinophils in vivo.

Recombinant human interleukin 5 is a selective activator of human eosinophil function.

Human rIL-5 was found to selectively stimulate morphological changes and the function of human eosinophils. This molecule is thus a prime candidate for the selective eosinophilia and eosinophil activation seen in disease.

Brief psychological interventions for psychiatric disorders in young people with long term physical health conditions: A systematic review and meta-analysis.

OBJECTIVES: Rates of psychiatric disorders are considerably elevated in young people with long term physical health conditions. Currently few children obtain effective mental health treatments in the context of long term physical health conditions, and ways to improve access to evidence-based mental health interventions are urgently needed. One approach is to deploy briefer, more economical, yet still evidence-based, treatments. The objective of this review was to evaluate the efficacy of brief interventions targeting psychiatric disorders in children and young people with long term physical health conditions. METHODS: Predefined terms relating to brief psychological interventions for psychiatric disorders in children with long term physical health conditions were used to search relevant databases. A systematic review and meta-analysis was carried out in accordance with the Cochrane guidelines. Two reviewers independently screened titles and abstracts, extracted the data and conducted risk of bias assessments. RESULTS: A total of 12 randomised controlled trials were found to meet the inclusion criteria of the review. Of those, three studies were suitable for meta-analysis. A large effect size in favour of brief cognitive behavioural therapy for anxiety was found (g = - 0.95, CI -1.49 to -0.041; p < .001) with non-significant moderate-substantial heterogeneity (I2 = 58%; p = .09). CONCLUSION: This review suggests there is preliminary evidence that brief interventions, based on cognitive behavioural principles, may benefit young people with an anxiety disorder in the context of a long term physical health condition. There was insufficient evidence to assess whether this held true for depression and disruptive behaviour.

Survival and quality of life for patients with COPD or asthma admitted to intensive care in a UK multicentre cohort: the COPD and Asthma Outcome Study (CAOS).

BACKGROUND: Non-invasive ventilation is first-line treatment for patients with acutely decompensated chronic obstructive pulmonary disease (COPD), but endotracheal intubation, involving admission to an intensive care unit, may sometimes be required. Decisions to admit to an intensive care unit are commonly based on predicted survival and quality of life, but the information base for these decisions is limited and there is some evidence that clinicians tend to be pessimistic. This study examined the outcomes in patients with COPD admitted to the intensive care unit for decompensated type II respiratory failure. METHODS: A prospective cohort study was carried out in 92 intensive care units and 3 respiratory high dependency units in the UK. Patients aged 45 years and older with breathlessness, respiratory failure or change in mental status due to an exacerbation of COPD, asthma or a combination of the two were recruited. Outcomes included survival and quality of life at 180 days. RESULTS: Of the 832 patients recruited, 517 (62%) survived to 180 days. Of the survivors, 421 (81%) responded to a questionnaire. Of the respondents, 73% considered their quality of life to be the same as or better than it had been in the stable period before they were admitted, and 96% would choose similar treatment again. Function during the stable pre-admission period was a reasonable indicator of function reported by those who survived 180 days. CONCLUSIONS: Most patients with COPD who survive to 180 days after treatment in an intensive care unit have a heavy burden of symptoms, but almost all of them-including those who have been intubated-would want similar intensive care again under similar circumstances.

Physical mapping of immune genes in the tammar wallaby (Macropus eugenii).

The tammar wallaby (Macropus eugenii) is a model marsupial that has recently had its genome sequenced to a depth of 2-fold coverage. Although this is a great resource for comparative genomic studies, information on gene location is essential if this resource is to be used to its full potential. In this study, tammar wallaby bacterial artificial chromosomes (BACs) containing key immune genes were isolated from the tammar wallaby BAC library. BACs containing T cell receptor (TCR) and immunoglobulin (Ig) genes were physically mapped using fluorescence in situ hybridisation (FISH) to tammar wallaby chromosomes. Congruence between the locations of these immune genes in the tammar wallaby genome, with those predicted from chromosome painting data, highlights the conservation of genomic context of these important immune genes in marsupials. The isolation and mapping of these key immune genes in the tammar wallaby will aid in the assembly of the recently sequenced light coverage genome and assignment of sequence to chromosomes.

Protein retention in yeast rough endoplasmic reticulum: expression and assembly of human ribophorin I.

The RER retains a specific subset of ER proteins, many of which have been shown to participate in the translocation of nascent secretory and membrane proteins. The mechanism of retention of RER specific membrane proteins is unknown. To study this phenomenon in yeast, where no RER-specific membrane proteins have yet been identified, we expressed the human RER-specific protein, ribophorin I. In all mammalian cell types examined, ribophorin I has been shown to be restricted to the membrane of the RER. Here we ascertain that yeast cells correctly target, assemble, and retain ribophorin I in their RER. Floatation experiments demonstrated that human ribophorin I, expressed in yeast, was membrane associated. Carbonate (pH = 11) washing and Triton X-114 cloud-point precipitations of yeast microsomes indicated that ribophorin I was integrated into the membrane bilayer. Both chromatography on Con A and digestion with endoglycosidase H were used to prove that ribophorin I was glycosylated once, consistent with its expression in mammalian cells. Proteolysis of microsomal membranes and subsequent immunoblotting showed ribophorin I to have assumed the correct transmembrane topology. Sucrose gradient centrifugation studies found ribophorin I to be included only in fractions containing rough membranes and excluded from smooth ones that, on the basis of the distribution of BiP, included smooth ER. Ribosome removal from rough membranes and subsequent isopycnic centrifugation resulted in a shift in the buoyant density of the ribophorin I-containing membranes. Furthermore, the rough and density-shifted fractions were the exclusive location of protein translocation activity. Based on these studies we conclude that sequestration of membrane proteins to rough domains of ER probably occurs in a like manner in yeast and mammalian cells.

2,4,5-trichlorophenoxyacetic acid causes behavioral effects in chickens at environmentally relevant doses.

Administration of the herbicide 2,4,5-trichlorophenoxyacetic acid to incubating chicken eggs alters behavior after hatching. Single doses, with no morphological effects, retard learning (lowest dose, 7 milligrams per kilogram of body weight) and increase general activity (27 milligrams per kilogram) and jumping (13 milligrams per kilogram). Day 15 of incubation is the most susceptible stage of development.

Preference for place of care and place of death in palliative care: are these different questions?

Place of death is at times suggested as an outcome for palliative care services. This study aimed to describe longitudinal preferences for place of care and place of death over time for patients and their caregivers. Longitudinal paired data of patient/caregiver dyads from a prospective unblinded cluster randomised control trial were used. Patients and caregivers were separately asked by the palliative care nurse their preference at that time for place of care and place of death. Longitudinal changes over time for both questions were mapped; patterns of agreement (patient and caregiver; and preference for place of death when last asked and actual placed of death) were analysed with kappa statistics. Seventy-one patient/caregiver dyads were analysed. In longitudinal preferences, preferences for both the place of care (asked a mean of >6 times) and place of death (asked a mean of >4 times) changed for patients (28% and 30% respectively) and caregivers (31% and 30%, respectively). In agreement between patients and caregivers, agreement between preference of place of care and preferred place of death when asked contemporaneously for patients and caregivers was low [56% (kappa 0.33) and 36% (kappa 0.35) respectively]. In preference versus actual place of death, preferences were met for 37.5% of participants for home death; 62.5% for hospital; 76.9% for hospice and 63.6% for aged care facility. This study suggests that there are two conversations: preference for current place of care and preference for care at the time of death. Place of care is not a euphemism for place of death; and further research is needed to delineate these. Patient and caregiver preferences may not change simultaneously. Implications of any mismatch between actual events and preferences need to be explored.

Tumor immunoprophylaxis in mice using glutaraldehyde-treated syngeneic tumor cells.

The ability of glutaraldehyde-treated tumor cells to induce protection against subsequent challenge has been studied in a syngeneic system. Two tumors have been tested in BALB/c mice. The first was a methylcholanthrene-induced tumor that has been maintained in serial passage for over a decade. The second was a spontaneous mammary adenoacanthoma that was tested at the third passage. The protection was found to consist of two components: (a) a specific immunological component; and (b) a nonspecific component observed when the immunizing and challenge dose were both given i.p. This nonspecificity, while possibly an element in tumor protective mechanisms, may confuse careful analytical studies of the immunogenic potential of tumor antigens.

Hierarchical Cluster Analysis of Three-Dimensional Reconstructions of Unbiased Sampled Microglia Shows not Continuous Morphological Changes from Stage 1 to 2 after Multiple Dengue Infections in Callithrix penicillata.

It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous.

Analysis of the 5' and 3'UTRs in the post-transcriptional regulation of the interleukin-5 gene.

Post-transcriptional regulation is emerging as an important control point in cytokine gene expression. However, the role that it plays in IL-5 gene expression is unclear with some conflicting reports. Here we investigate the importance of post-transcriptional regulation and the role of the 5' and 3' untranslated regions (UTRs) in mIL-5 gene expression. To do this, IL-5 expression from a panel of cDNA constructs was compared. We found it essential to remove the 5' synthetic oligonucleotide tails, introduced during the cloning of the mIL-5 cDNA, when studying IL-5 expression. The presence of these oligo(G) tails acted as potent inhibitors of translation of both SV40 and SP6 transcripts. Furthermore, the length of the tails was found to be critical to the translational efficiency. Taking this into account, we found no evidence to suggest that IL-5 is regulated at the level of mRNA stability or translation efficiency by either the 5' or 3'UTR. These results suggest that post-transcriptional control is not a major factor regulating IL-5 expression.

Interleukin-5 binds to heparin/heparan sulfate. A model for an interaction with extracellular matrix.

Interleukin-5 (IL-5) is the major cytokine regulating eosinophil production. In allergic disease tissue damage is primarily caused by eosinophils. Heparan sulfate proteoglycans are components of the bone marrow stroma, which supports hemopoietic cell differentiation and proliferation. We show that at low IL-5 concentrations heparan sulfate enhances the proliferation of an IL-5-dependent cell line. To investigate a mechanism for this effect we used an artificial proteoglycan to establish an enzyme-linked immunosorbent assay for the binding of heparin to proteins. Using this assay we demonstrate that IL-5 binds to heparin. The IL-5/heparin interaction is inhibited by ethylenediaminetetraacetate and enhanced by low concentrations of zinc ions. IL-5 interacts with iduronic acid containing glycosaminoglycans, and heparan sulfate preparations that have numerous N-sulfated domains per chain are especially efficient at inhibiting heparin binding. Both IL-5/heparin binding and the synergistic effect of IL-5 and heparan sulfate on cell proliferation were inhibited by an anti-IL-5 monoclonal antibody. These data suggest that the binding of IL-5 to heparan sulfate modulates IL-5 activity.

Mutated interleukin-5 monomers are biologically inactive.

Interleukin-5 contains only two cysteine residues both of which appear to be involved in the dimerisation of the molecule to form a disulphide-linked homodimer (Minamitake et al., J. Biochem. 107, 292-297, 1990). However, it remains unclear whether this linkage is necessary for the bioactivity of this cytokine. Site-directed mutagenesis was used to produce amino acid substitutions of either or both of the cysteines. The mutant proteins were all biologically inactive monomers, however when the two single mutant constructs were co-transfected, biologically active IL5 was produced. This is consistent with the dimer forming in a head-to-tail configuration.

Identification of two novel palindromic regulatory elements in the murine interleukin-5 promoter.

Interleukin-5 has remarkable specificity for the eosinophil lineage. This fact, combined with the biological specificity of eosinophilia suggests tight and independent regulation of IL-5 expression. Here we report two novel palindromic regulatory elements (PRE) which contain positive regulatory motifs (PRM) that control transcription of the murine IL-5 gene. The first element, mPRE1-IL5 (-79 to -90) contains the mPRM1 at positions -87 to -89 which operates as a positive regulatory element with mutation of this motif resulting in a 64% decrease in gene activity. Gene expression was reduced by 67% when a similar mutation was introduced into the mPRM2 (-467 to -469) of mPRE2-IL5 (-459 to -470). Both elements specifically bind proteins from EL4-23 cell nuclear extracts forming constitutive DNA-protein complexes. EMSA experiments utilising mutated mPRE-IL5 oligonucleotides indicate that in both elements, the mPRMs are essential for protein binding.

Infection of IL5 transgenic mice with Mesocestoides corti induces very high levels of IL5 but depressed production of eosinophils.

Transgenic mice expressing interleukin 5 (IL5) have been demonstrated to show a lifelong high level eosinophilia. These mice were produced using a construct in which the dominant control region (DCR) of the human CD2 gene was ligated to a 10-kb fragment containing the mouse IL5 gene. The construct allows the expression of the IL5 gene under the control of its own promoter, but the DCR ensures constitutive expression by all T cells. Infection of these transgenic mice with Mesocestoides corti, which is itself a potent inducer of eosinophilia, increases serum IL5 to very high levels. This demonstrates that the transgenes retain inducibility, which is a feature of the endogenous gene. However, despite the high levels of IL5, the numbers of eosinophils in the blood, marrow, and spleen decrease during the period 1-4 weeks after infection. Furthermore, there is a decrease in eosinophil precursors, as assessed by the capacity of bone marrow to produce eosinophils in culture. After this decrease eosinophils return to their previous high levels, although the levels of IL5 remain high. These results suggest that a control mechanism is operating to limit the numbers of eosinophils produced. This control appears to act at the level of the precursor production and may not be directly related to the high levels of IL5.