Acute effect of hemodialysis on serum levels of the proinflammatory cytokines

Chronic inflammation is a common feature of end-stage renal disease, which carries a heightened risk of atherosclerosis and other co-morbid conditions. Dialysis treatment per se can bring additional risk factors for inflammation, such as increased risk of local graft and fistula infections, impure dialysate or bio-incompatible membranes. Our study was designed to determine whether a hemodialysis session leads to an acute substantial alteration in the plasma levels of the proinflammatory cytokines interleukin (IL)-6, IL-1beta and tumor necrosis factor (TNF)-alpha, the T-lymphocyte activation factor soluble IL-2 receptor (sIL-2R), and an inflammation mediator and chemotactic granulocyte factor, IL-8, in end-stage renal disease patients receiving chronic intermittent HD. In this study, 21 (12 male/nine female) patients undergoing chronic hemodialysis were enrolled. The acute effect of a hemodialysis session on serum cytokine concentrations was assessed by comparison of pre-hemodialysis and post-hemodialysis determinations. Serum IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha levels were determined with chemiluminescence enzyme immunometric assays. A significant difference was not observed for IL-1beta, IL-6, TNF-alpha, and sIL-2R concentrations in pre-hemodialysis and post-hemodialysis specimens (p>0.05). Serum median (25th-75th percentiles) IL-8 concentration was 69.4 (34.9-110.3) pg/ml before hemodialysis, and decreased to 31.5 (18.0-78.8) pg/ml following hemodialysis (p: 0.006). Clearance of IL-8 increased by 0.47+/-0.08 pg/ml for each unit increase in pre-dialysis IL-8 (p<0.001) and decreased by 5.63+/-2.59 pg/ml for each unit increase in pre-dialysis urea mmol/l (p<0.05). In conclusion, the results of our study demonstrate that a hemodialysis session markedly decreases IL-8 concentration, which is significantly affected by pre-dialysis concentrations, indicating that removal of IL-8 is a concentration gradient-dependent action, but does not change the serum levels of IL-1beta, sIL-2R, IL-6, and TNF-alpha, underlining importance of the structural characteristics of the molecules.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.     

The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.     

Adenosine triphosphate alters the selenite-induced contracture and negative inotropic effect on cardiac muscle contractions

Selenium is known to play an important role in the physiology of many different cell types and extracellular application of selenite causes cellular dysfunction in many different types of tissues. In a previous study, we have shown that in rat ventricles, sodium selenite (≥1 mM) caused an increase in the resting tension and a decrease in contractile force, in a time-dependent manner. In the present study, we have shown that sodium selenite caused a contracture state both in Langendorff perfused hearts and isolated papillary muscles. We also showed that the application of extracellular ATP (0.1 mM) markedly reduced this detrimental effect of sodium selenite on ventricular contraction in Langendorff perfused hearts and delayed it in isolated papillary muscle preparations. In contrast, isoproterenol (0.1 μM) did not seem to influence this action of sodium selenite in papillary muscle preparations. Possible reasons for this protective effect of ATP to selenite-induced contracture are also discussed.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Purification of glucose 6-phosphate dehydrogenase from chicken erythrocytes. investigation of some kinetic properties

Glucose 6-phosphate dehydrogenase (G6PD) was purified from chicken erythrocytes, and some characteristics of the enzyme were investigated. The purification procedure was composed of three steps: hemolysate preparation, ammonium sulfate precipitation, and 2',5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 20.862 EU/mg proteins, was purified with a yield of 54.68% and 9,150-fold. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, Ki values and the type of inhibition were determined by means of Line-Weaver-Burk graphs obtained for such inhibitors as ATP, ADP, NADH, and NADPH.     

Neutrophil chemotaxis in linear and complex gradients of interleukin-8 formed in a microfabricated device

Although a wealth of knowledge about chemotaxis has accumulated in the past 40 years, these studies have been hampered by the inability of researchers to generate simple linear gradients instantaneously and to maintain them at steady state. Here we describe a device microfabricated by soft lithography and consisting of a network of microfluidic channels that can generate spatially and temporally controlled gradients of chemotactic factors. When human neutrophils are positioned within a microchannel, their migration in simple and complex interleukin-8 (IL-8) gradients can be tested. The cells exhibit strong directional migration toward increasing concentrations of IL-8 in linear gradients. Neutrophil migration halts abruptly when cells encounter a sudden drop in the chemoattractant concentration to zero ("cliff" gradient). When neutrophils are challenged with a gradual increase and decrease in chemoattractant ("hill" gradient), however, the cells traverse the crest of maximum concentration and migrate further before reversing direction. The technique described in this paper provides a robust method to investigate migratory cells under a variety of conditions not accessible to study by earlier techniques.     

The effect of high dose digoxin on cytokines in healthy dogs

BACKGROUND: Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta are pro-inflammatory cytokines, causing myocardial dysfunction and a negative inotropic effect. The drugs used to treat heart failure affect the production of cytokines. Digoxin, on which this study was focused, is one of the drugs for the treatment of heart failure. AIM: The present study was designed to examine the early effects of high doses of digoxin on the production of cytokines in healthy dogs. METHODS: Digoxin was given parenterally to dogs at 0.15 mg/kg. IL-1beta and TNF-alpha production and levels of digoxin in the serum were measured 0, 12, 24, 48, and 72 h following administration of digoxin. RESULTS: As the levels of serum digoxin taken at 12, 24, 48, and 72 h of administration were considered significantly high compared with preceding values (p < 0.001), no notable change in serum IL-1beta and TNF-alpha levels was observed. CONCLUSIONS: These results suggest that high doses of digoxin do not cause a significant cytokine production in heart muscle in the early phase.