Co-activation of JAK/STAT and p38 MAPK pathways by type I interferon enhances apoptosis in human basophils

It has previously been published that interferon-α (type I IFN) improves clinical symptoms of asthma patients. Since human basophils are major inflammatory cells in maintaining chronic allergic asthma we investigate whether type I IFN affect human blood basophils. Furthermore, previous studies have shown that spontaneous apoptosis of human basophils is slow due to constitutive expression of anti-apoptotic BCL-2 family members. In addition, IL-3 exceptionally promotes survival of basophils by enhancing constitutive expression of BCL-2 family members and by inducing de-novo expression of Pim-1 kinase. Thus, we also assessed whether type I IFN might overcome IL-3-induced survival of human basophils. Our data show that type I IFN enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or type II IFN treated cells. Furthermore, we demonstrate that both type I IFN and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. Analyses of signaling pathways reveal that type I IFN promote prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of type I IFN is abolished. On the other hand, type I IFN do not reduce IL-3-induced de novo expression of Pim-1 and BCL-2. This is in line with our observation that IL-3-induced survival is dominant over type I IFN-enhanced apoptosis. In addition, phosphorylation of p38 MAPK in type I IFN treated cells is comparable to non-treated cells. Particularly however, inhibition of this p-p38 activity abrogates apoptosis as well. We conclude that type I IFN-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38 MAPK pathways. Our study identifies a so far unknown effect of type I IFN and may explain the improved clinical symptoms of asthma patients treated with type I IFN.

Interferon-α antagonizes IL-3-mediated immunoregulatory functions of human basophils

Human basophils are major inflammatory cells in maintaining chronic allergic asthma. It has been published that interferon-α (IFN-α) improves clinical symptoms of asthma patients. In contrast, IL-3 exacerbates airway inflammation by inducing IL-4, IL-8 and IL-13 secretion from human basophils thus regulating their immunoregulatory functions. Furthermore, IL-3 exceptionally promotes survival of basophils. Here, we assessed cellular response of human basophils treated with IFN-α alone or in combination with IL-3. Our data show that IFN-α enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or IFN-γ treated cells. Furthermore, we demonstrate that both IFN-α and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. IFN-α inhibits IL-3-induced survival to a minor degree. Particularly however, it suppresses IL-3-induced de-novo production of IL-8 and IL-13 up to 80%. In contrast, the production of IL-4 is not affected. Analyses of signaling pathways reveal that IFN-α promotes prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of IFN-α is abolished. Although the phosphorylation of p38-MAPK in IFN-α-treated cells is comparable to non-treated cells, inhibition of p-p38 activity abrogates IFN-α-enhanced apoptosis as well. We conclude that IFN-α-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38-MAPK pathways. Our study identifies IFN-α as a novel inhibitor of IL-3-induced IL-8 and IL-13 production of human basophils. Taken together our study may explain the improved clinical symptoms of asthma patients treated with IFN-α.

Solid phase phosphorus geochemistry in surface sediments of sediment cores GeoB3718-4, GeoB3702-2 and GeoB3707-4

In this study we investigate benthic phosphorus cycling in recent continental margin sediments at three sites off the Namibian coastal upwelling area. Examination of the sediments reveals that organic and biogenic phosphorus are the major P-containing phases preserved. High Corg/Porg ratios just at the sediment surface suggest that the preferential regeneration of phosphorus relative to that of organic carbon has either already occurred on the suspension load or that the organic matter deposited at these sites is already rather refractory. Release of phosphate in the course of benthic microbial organic matter degradation cannot be identified as the dominating process within the observed internal benthic phosphorus cycle. Dissolved phosphate and iron in the pore water are closely coupled, showing high concentrations below the oxygenated surface layer of the sediments and low concentrations at the sediment-water interface. The abundant presence of Fe(III)-bound phosphorus in the sediments document the co-precipitation of both constituents as P-containing iron (oxyhydr)oxides. However, highly dissolved phosphate concentrations in pore waters cannot be explained, neither by simple mass balance calculations nor by the application of an established computer model. Under the assumption of steady state conditions, phosphate release rates are too high as to be balanced with a solid phase reservoir. This discrepancy points to an apparent lack of solid phase phosphorus at sediment depth were suboxic conditions prevail. We assume that the known, active, fast and episodic particle mixing by burrowing macrobenthic organisms could repeatedly provide the microbially catalyzed processes of iron reduction with authigenic iron (oxyhydro)oxides from the oxic surface sediments. Accordingly, a multiple internal cycling of phosphate and iron would result before both elements are buried below the iron reduction zone.

Porewater and solid-phase phosphorus geochemistry in surface sediments of sediment core GeoB9510-3, GeoB9518-4 and GeoB9519-6

Despite intensive research on the different domains of the marine phosphorus (P) cycle during the last decades, frequently discussed open questions still exist especially on controlling factors for the benthic behaviour of P and its general distribution in sediment-pore water systems. Steady state or the internal balance of all relevant physical and (bio)geochemical processes are amongst the key issues. In this study we present and discuss an extended data set from surface sediments recovered from three locations on the NW African continental slope. Pore water data and results from sequential sediment extractions give clear evidence to the well-known close relationship between the benthic cycles of P and iron. Accordingly, most of the dissolved phosphate must have been released by microbially catalyzed reductive dissolution of iron (oxhydr)oxides. However, rates of release and association of P and iron, respectively, are not directly represented in profiles of element specific sediment compositions. Results from steady-state based transport-reaction modelling suggest that particle mixing due to active bioturbation, or rather a physical net downward transport of P associated to iron (oxyhydr)oxides, is an essential process for the balance of the inspected benthic cycles. This study emphasizes the importance of balancing analytical data for a comprehensive understanding of all processes involved in biogeochemical cycles.

Identification of genes required for Drosophila eye development using a phenotypic enhancer-trap

A novel method of P-element mutagenesis is described for the isolation of mutants affecting the development of the Drosophila compound eye. It exploits the interaction between the Bride of Sevenless (Boss) ligand and the Sevenless (Sev) receptor tyrosine kinase that triggers the formation of the UV-sensitive photoreceptor neuron, R7. Transposition of a boss cDNA transgene, in an otherwise boss mutant background, was used as a “phenotypic trap” in live flies to identify enhancers expressed during a narrow time window in eye development. Using a rapid behavioral screen, more than 400,000 flies were tested for restoration of R7. Some 1,800 R7-containing flies were identified. Among these, 21 independent insertions with expression of the boss reporter gene in the R8 cell were identified by a external eye morphology and staining with an antibody against Boss. Among 900 lines with expression of the boss reporter gene in multiple cells assessed for homozygous mutant phenotypes, insertions in the marbles, glass, gap1, and fasciclin II genes were isolated. This phenotypic enhancer-trap facilitates (i) the isolation of enhancer-traps with a specific expression pattern, and (ii) the recovery of mutants disrupting development of specific tissues. Because the temporal and tissue specificity of the phenotypic trap is dependent on the choice of the marker used, this approach can be extended to other tissues and developmental stages.

Early physiological abnormalities after simian immunodeficiency virus infection

Central nervous system (CNS) damage and dysfunction are devastating consequences of HIV infection. Although the CNS is one of the initial targets for HIV infection, little is known about early viral-induced abnormalities that can affect CNS function. Here we report the detection of early physiological abnormalities in simian immunodeficiency virus-infected monkeys. The acute infection caused a disruption of the circadian rhythm manifested by rises in body temperature, observed in all five individuals between 1 and 2 weeks postinoculation (p.i.), accompanied by a reduction in daily motor activity to 50% of control levels. Animals remained hyperthermic at 1 and 2 months p.i. and returned to preinoculation temperatures at 3 months after viral inoculation. Although motor activity recovered to baseline values at 1 month p.i., activity levels then decreased to approximately 50% of preinoculation values over the next 2 months. Analysis of sensory-evoked responses 1 month p.i. revealed distinct infection-induced changes in auditory-evoked potential peak latencies that persisted at 3 months after viral inoculation. These early physiological abnormalities may precede the development of observable cognitive or motor deficiencies and can provide an assay to evaluate agents to prevent or alleviate neuronal dysfunction.

Cloning and Characterization of peter pan, a Novel Drosophila Gene Required for Larval Growth

We identified a new Drosophila gene, peter pan (ppan), in a screen for larval growth–defective mutants. ppan mutant larvae do not grow and show minimal DNA replication but can survive until well after their heterozygotic siblings have pupariated. We cloned the ppan gene by P-element plasmid rescue. ppan belongs to a highly conserved gene family that includes Saccharomyces cerevisiae SSF1 and SSF2, as well as Schizosaccharomyces pombe, Arabidopsis, Caenorhabditis elegans, mouse, and human homologues. Deletion of both SSF1 and SSF2 in yeast is lethal, and depletion of the gene products causes cell division arrest. Mosaic analysis of ppan mutant clones in Drosophila imaginal disks and ovaries demonstrates that ppan is cell autonomous and required for normal mitotic growth but is not absolutely required for general biosynthesis or DNA replication. Overexpression of the wild-type gene causes cell death and disrupts the normal development of adult structures. The ppan gene family appears to have an essential and evolutionarily conserved role in cell growth.

Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/− mice that exhibit defective NK cell development. In the Id2−/− FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/− FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/− fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/− and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Pharmacokinetic considerations relating to tacrolimus dosing in the elderly

Relaxation of the upper age limits for solid organ transplantation coupled with improvements in post-transplant survival have resulted in greater numbers of elderly patients receiving immunosuppressant drugs such as tacrolimus. Tacrolimus is a potent agent with a narrow therapeutic window and large inter- and intraindividual pharmacokinetic variability. Numerous physiological changes occur with aging that could potentially affect the pharmacokinetics of tacrolimus and, hence, patient dosage requirements. Tacrolimus is primarily metabolised by cytochrome P450 (CYP) 3A enzymes in the gut wall and liver. It is also a substrate for P-glycoprotein, which counter-transports diffused tacrolimus out of intestinal cells and back into the gut lumen. Age-associated alterations in CYP3A and P-glycoprotein expression and/or activity, along with liver mass and body composition changes, would be expected to affect the pharmacokinetics of tacrolimus in the elderly. However, interindividual variation in these processes may mask any changes caused by aging. More investigation is needed into the impact aging has on CYP and P-glycoprotein activity and expression. No single-dose, intense blood-sampling study has specifically compared the pharmacokinetics of tacrolimus across different patient age groups. However, five population pharmacokinetic studies, one in kidney, one in bone marrow and three in liver transplant recipients, have investigated age as a co-variate. None found a significant influence for age on tacrolimus bioavailability, volume of distribution or clearance. The number of elderly patients included in each study, however, was not documented and may have been only small. It is likely that inter- and intraindividual pharmacokinetic variability associated with tacrolimus increase in elderly populations. In addition to pharmacokinetic differences, donor organ viability, multiple co-morbidity, polypharmacy and immunological changes need to be considered when using tacrolimus in the elderly. Aging is associated with decreased immunoresponsiveness, a slower body repair process and increased drug adverse effects. Elderly liver and kidney transplant recipients are more likely to develop new-onset diabetes mellitus than younger patients. Elderly transplant recipients exhibit higher mortality from infectious and cardiovascular causes than younger patients but may be less likely to develop acute rejection. Elderly kidney recipients have a higher potential for chronic allograft nephropathy, and a single rejection episode can be more devastating. There is a paucity of information on optimal tacrolimus dosage and target trough concentration in the elderly. The therapeutic window for tacrolimus concentrations may be narrower. Further integrated pharmacokinetic-pharmaco-dynamic studies of tacrolimus are required. It would appear reasonable, based on current knowledge, to commence tacrolimus at similar doses as those used in younger patients. Maintenance dose requirements over the longer term may be lower in the elderly, but the increased variability in kinetics and the variety of factors that impact on dosage suggest that patient care needs to be based around more frequent monitoring in this age group.

Hepatic pharmacokinetics of propranolol in rats with adjuvant-induced systemic inflammation

Systemic inflammation is known to affect drug disposition in the liver. This study sought to relate and quantitate changes in hepatic pharmacokinetics of propranolol with changes in hepatic architecture and physiology in adjuvant-treated rats. Transmission electron microscopy was used to assess morphological changes in mitochondria and lysosomes of adjuvant-treated rat livers. The disposition of propranolol was assessed in the perfused rat liver using the multiple indicator dilution technique. Hepatic extraction and mean transit time were determined from outflow-concentration profiles using a nonparametric method. Kinetic parameters were derived from a two-phase physiologically based organ pharmacokinetic model. Possible relationships were then explored between the changes in hepatic drug disposition and cytochrome P-450 activity and iron concentration. Adjuvant treatment induced the appearance of mitochondrial inclusions/tubularization and irregularly shaped lysosomes in rat livers. Livers from adjuvant-treated rats had (relative to normal) significantly higher alpha(1)-acid glycoprotein (orosomucoid) and iron tissue concentrations but lower cytochrome P-450 content. The hepatic extraction, metabolism, and ion trapping of propranolol were significantly impaired in adjuvant-treated rats and could be correlated with altered iron store and cytochrome P-450 activity. It is concluded that adjuvant-induced systemic inflammation alters hepatocellular morphology and biochemistry and consequently influences hepatic disposition of propranolol.

Investigating the modulation of oral drug absorption using in vitro models

Dipeptides can be absorbed into cells via the dipeptide transporter (which also transported tripeptides and dipeptide derivatives). The optimum conditions for measuring the inhibition of Gly-Pro uptake in Caco-2 cells were identified. A number of structure-activity relationships were identified. These included the effects of increasing the amino-acid chain-length, and the presence of a thiol or hydroxyl group in the side-chain increased IC50 while the presence of a hydroxyl group did not. The benzyl esters had lower or equal IC50 values compared to the parent dipeptides while the methyl esters had higher values. These results indicated that while molecular properties did affect IC50, the size, charge and composition of three particular groups caused the most significant effects, supporting the structure-activity relationship identified. An assay was developed using calcein-AM to show the inhibition of p-glycoprotein activity. There was no significant change due to the presence of mannitol but there was in the presence of clyclosporin A (p<0.01). Incubating the cells with the test solution for 30 minutes before the addition of the ester resulted in a significant (p<0.001) difference. The assay was specific for p-glycoprotein, as the presence MRP inhibitors had no effect (p>0.05). The modified protocol allowed the identification of p-glycoprotein inhibitors quickly and simply using a cell suspension of unmodified cells. The clinically relevant buffering of grapefruit juice to pH 7 led to a four-fold increase in intracellular calcein and hence significant inhibition of p-glycoprotein. Buffered orange and lemon juices had no effect on the assay. Flavone derivatives had previously been found to be inhibitors of CYP3A4 yet neither naringin nor naringenin had any significant effect at concentrations found in grapefruit juice. Of the other (non-grapefruit) flavone derivatives tested, hesperidin, found in orange juice, had no significant effect, kaempferol and rutin also had no effect while genistein significantly inhibited p-glycoprotein (results that support previous studies). Hydroxycinnamic acids had no effect on p-glycoprotein. Studies on other compounds found that the balance between inhibiting p-glycoprotein and disrupting cell membranes depends on the compound containing an oxygen atom and the size of the negative charge on it, as well as three-dimensional arrangement of the atoms.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

exl protein specifically binds BLE1, a bicoid mRNA localization element, and is required for one phase of its activity.

Localization of mRNAs, a crucial step in the early development of some animals, has been shown to be directed by cis-acting elements that presumably interact with localization factors. Here we identify a protein, exl, that binds to BLE1, an RNA localization element from the Drosophila bicoid mRNA. Using mutations in BLE1, we demonstrate a correlation between in vitro exl binding and one phase of in vivo localization directed by BLE1, implicating exl in that localization event. Furthermore, the same phase of localization is disrupted in exuperantia mutants, suggesting that exl and exuperantia proteins interact. Identification of a protein that binds specifically to an mRNA localization element and acts in mRNA localization opens the way for a biochemical analysis of this process.

Information from managing information systems as a fundamental element to support decision-making in hospitals

This article deals with the activity of defining information of hospital systems as fundamental for choosing the type of information systems to be used and also the organizational level to be supported. The use of hospital managing information systems improves the user`s decision -making process by allowing control report generation and following up the procedures made in the hospital as well.

Brazilian Propolis: Seasonal Variation of the Prenylated p-Coumaric Acids and Antimicrobial Activity

Brazilian green propolis, which is used in food and beverages to improve health and to prevent diseases, demostrates antioxidant, antimutagenic, and antimicrobial activities. Most biological activities are thought to be related to the high levels of drupanin, artepillin C, and baccharin, which are compounds also present in Baccharis dracunculifolia D.C. (Asteraceae). Since propolis chemical composition depends on the region and the period of collection, as well as its plant origin, the effect of seasonal variation on the both content of prenylated p-coumaric acids and in vitro antimicrobial activity of Brazilian propolis from four different sites, was performed. The results showed that MIC values ranged from 100 to 300 mu g/mL against both Staphylococcus aureus and Kocuria rhizophila, while none of the propolis samples was active against Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. HPLC analysis showed that the content of drupanin, artepillin C, and baccharin varied throughout the year, as well as among the different study sites. Also, it is suggested that Baccharis dracunculifolia is the main botanical source of Brazilian propolis in sites I and 2, while in sites 3 and 4, other plant species are also used by bees to produce propolis. All the evaluated propolis samples exhibited similar antibacterial activity, but different contents of prenylated p-coumaric acids throughout the year.