Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.     

Production, purification, and luminometric analysis of recombinant Saccharomyces cerevisiae MET3 adenosine triphosphate sulfurylase expressed in Escherichia coli

ATP sulfurylase cDNA from MET3 on chromosome X of Saccharomyces cerevisiae was amplified and cloned, and recombinant ATP sulfurylase was expressed in Escherichia coli. The synthesis of ATP sulfurylase was directed by an expression system that employs the regulatory genes of the luminous bacterium Vibrio fischeri. A soluble, biologically active form was purified to electrophoretic homogeneity from lysates of recombinant E. coli by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The specific activity of the purified enzyme was estimated to 140 U/mg. The apparent molecular mass of the recombinant enzyme was determined by gel filtration to be 470 kDa, which indicates that the active enzyme is an octamer of identical subunits (the molecular mass of a single subunit is 59.3 kDa). The ATP sulfurylase activity was monitored in real time by a very sensitive bioluminometric method.     

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.     

Indirect effects of lemming cycles on sandpiper dynamics: 50 years of counts from southern Sweden

The bird-lemming hypothesis postulates that breeding success of tundra-nesting geese and waders in Siberia follows the cyclic pattern of lemming populations, as a result of predators switching from lemmings to birds when the lemming population crashes. We present 50 years of data on constant-effort catches of red knot Calidris canutus and curlew sandpiper C. ferruginea at an autumn migratory stopover site (Ottenby) at the Baltic Sea, supplemented with literature data on winter censuses of dark-bellied brent goose Branta b. bernicla and white-fronted goose Anser albifrons in northwestern Europe, and waders in Germany and Southern Africa. Number and proportion of juveniles in these bird populations (both our own and literature data) were compared with an index of predation pressure (calculated from the abundance of lemmings on the Taimyr peninsula), and climate indices for the North Eurasia and the North Atlantic regions. The index of predation pressure correlated significantly with the number of juveniles of red knot and curlew sandpiper, but not with number of adults. Also, this index correlated with the reproductive performance of geese and waders reported in the literature. Fourier analysis revealed a significant deviation from random noise with the maximum spectral density at the period length of 3 years for number of juvenile red knots and curlew sandpipers captured at Ottenby, abundance of lemmings, reproduction in arctic fox Alopex lagopus, and reproductive performance in geese on the Siberian tundra. Also, the date of passage at Ottenby for adult red knot and curlew sandpiper showed a spectral density peak at a period length of 3 years, the latter species also showing a peak at a period length of 5-6 years. Passage dates for adult red knot and curlew sandpiper were earlier in years of high predation pressure compared with years of low predation pressure. The fluctuations in reproductive success of the studied Siberian goose and wader species appear to be primarily influenced by biotic factors in the breeding area, rather than by abiotic factors, such as climate oscillations. Annual variations in migratory arctic bird populations may have far reaching effects in habitats along their migration routes and in their wintering areas. We suggest a link between lemming cyclicity in the Northern Hemisphere and predation pressure on Southern Hemisphere benthos, in which the signal is carried between continents by long distance migrating waders.     

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus

AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.     

Speciation in vanadium bioinorganic systems. 6. Speciation study of aqueous peroxovanadates, including complexes with imidazole

Using a combination of potentiometry (glass electrode) and quantitative 51V NMR spectroscopy, the full speciation in the vanadate-peroxide and vanadate-peroxide-imidazole systems was determined in the pH range 1-10 (0.150 M Na(Cl) medium, 25 degrees C). Using the computer program LAKE, the pKa value of imidazole and the formation constants for 10 peroxovanadate species and also for three more species where a single imidazole moiety is also bound, have been calculated. The experimental data show a good fit to the calculated speciation model, even for the less abundant species. The species are either monomeric or dimeric in vanadium, and four resonances of the dimeric species have been unambiguously assigned via 2D 51V NMR. Diperoxovanadates are the favoured species at pH 2-10, when sufficient peroxide is present. Imidazole is found to bind strongly to them at pH 6-9. The equilibrium conditions are illustrated in distribution diagrams.     

Development of an internally controlled PCR assay for broad range detection of bacteria in platelet concentrates

A real-time PCR assay based on the 16S rRNA gene was optimized for the detection of a broad range of bacteria in plasma and platelet concentrates (PC). A lambda phage internal control was constructed and implemented in the assay, which made it suitable for diagnostic use. Spiking studies in plasma and PCs were performed to determine the analytical sensitivity of the assay. Thirty three colony forming units (CFU)/ml of E. coli and 72 CFU/ml of Staphylococcus epidermidis could be detected in plasma, and 97 CFU/ml of S. epidermidis in PCs. The assay detected all bacteria relevant for bacterial contamination of PCs. The short turn around time of the assay made it suitable for testing PCs for bacterial contamination prior to transfusion.