Highly biased type 1 immune responses in mice deficient in LFA-1 in Listeria monocytogenes infection are caused by elevated IL-12 production by granulocytes

LFA-1 (CD11a/CD18) plays a key role in various inflammatory responses. Here we show that the acquired immune response to Listeria monocytogenes is highly biased toward type 1 in the absence of LFA-1. At the early stage of listeriosis, numbers of IFN-gamma producers in the liver and spleen of LFA-1(-/-) mice were markedly increased compared with heterozygous littermates and Valpha14(+)NKT cell-deficient mice, and NK cells were major IFN-gamma producers. Numbers of IL-12 producers were also markedly elevated in LFA-1(-/-) mice compared with heterozygous littermates, and endogenous IL-12 neutralization impaired IFN-gamma production by NK cells. Granulocyte depletion diminished numbers of IL-12 producers and IFN-gamma-secreting NK cells in the liver of LFA-1(-/-) mice. Granulocytes from the liver of L. monocytogenes-infected LFA-1(-/-) mice were potent IL-12 producers. Thus, in the absence of LFA-1, granulocytes are a major source of IL-12 at the early stage of listeriosis. We assume that highly biased type 1 immune responses in LFA-1(-/-) mice are caused by increased levels of IL-12 from granulocytes and that granulocytes play a major role in IFN-gamma secretion by NK cells. In conclusion, LFA-1 regulates type 1 immune responses by controlling prompt infiltration of IL-12-producing granulocytes into sites of inflammation.     

Glutamate modifies ion conduction and voltage-dependent gating of excitatory amino acid transporter-associated anion channels

Excitatory amino acid transporters (EAATs) mediate two distinct transport processes, a stoichiometrically coupled transport of glutamate, Na+, K+, and H+, and a pore-mediated anion conductance. We studied the anion conductance associated with two mammalian EAAT isoforms, hEAAT2 and rEAAT4, using whole-cell patch clamp recording on transfected mammalian cells. Both isoforms exhibited constitutively active, multiply occupied anion pores that were functionally modified by various steps of the Glu/Na+/H+/K+ transport cycle. Permeability and conductivity ratios were distinct for cells dialyzed with Na(+)- or K(+)-based internal solution, and application of external glutamate altered anion permeability ratios and the concentration dependence of the anion influx. EAAT4 but not EAAT2 anion channels displayed voltage-dependent gating that was modified by glutamate. These results are incompatible with the notion that glutamate only increases the open probability of the anion pore associated with glutamate transporters and demonstrate unique gating mechanisms of EAAT-associated anion channels.     

Adaptive reproductive variation along a pollution gradient in a wolf spider

When populations are exposed to environmental pollutants, growth and reproduction might be strongly reduced due to an increased detoxification effort. Sublethal metal pollution is therefore to be expected to cause the same selection pressure as a low resource habitat and might alter the reproductive strategy. Optimality models of life history theory predict that when resource availability is reduced, growth and reproductive output are reduced and that the release of fewer but larger propagules will be favoured. This was tested by applying a life history model to reproductive trait measurements in six populations of the wolf spider Pirata piraticus in which the assumptions of the model are satisfied. Internal Cd, Cu and Zn body burden were strongly correlated with each other, and differed strongly between the populations, indicating consistently differing metal exposure at the different sites. Pb levels were extremely variable within each population and did not differ between the populations. Females from populations with high concentrations of the first three heavy metals showed a strongly reduced reproductive output and fecundity, indicating a high reduction in resource availability due to detoxification processes. Egg size in contrast was negatively correlated with fecundity and reproductive output and as a consequence positively related with internal metal burden. Our results are thus in strong agreement with the predictions of the optimality models and confirm the benefits of a larger propagule size when resource availability is reduced.     

Coronary perfusion and muscle lengthening increase cardiac contraction: different stretch-triggered mechanisms

An increase in coronary perfusion, transversal stretch of the myocardium, increases developed force (F(dev)) (Gregg effect) through activation of stretch-activated ion channels (SACs). Lengthening of the muscle, longitudinal stretch of the myocardium, causes an immediate increase in F(dev) followed by a slow F(dev) increase (Anrep effect). In isometrically contracting perfused papillary muscles of Wistar rats, we investigated whether both effects were based on similar stretch-induced mechanisms by measuring F(dev) and intracellular Ca(2+) concentration ([Ca(2+)](i)) after a muscle length increase from 85% to 95% L(max) (length at which maximal isometric force develops) at low and high coronary perfusion before and after inhibition of SACs with gadolinium (10 micromol/l Gd(3+)). The increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect was of similar magnitude as the Anrep effect (from 3.5 +/- 0.8 to 3.9 +/- 1.2 mN/mm(2) and from 3.0 +/- 0.7% to 3.8 +/- 0.9% normalized [Ca(2+)](i), means +/- SE). SAC blockade completely blunted the increase of F(dev) and peak [Ca(2+)](i) by the Gregg effect; however, it did not affect the Anrep effect. The slow force response, but not the calcium response, was augmented by an increase in coronary perfusion. Therefore, increased coronary perfusion, transversal stretch of the myocardium, and muscle lengthening, longitudinal stretch of the myocardium, increase myocardial contraction in the rat through different stretch-triggered mechanisms.     

Tissue distribution of cytosolic beta-elimination reactions of selenocysteine Se-conjugates in rat and human

Selenocysteine Se-conjugates (e.g. methylselenocysteine) have been shown to be potent chemopreventive and chemoprotective agents, and inducers of apoptosis. Although the mechanism of action remains to be elucidated, beta-elimination of these compounds by beta-lyase enzymes into corresponding selenols, pyruvate and ammonia is thought to be critical. This study describes in vitro beta-lyase activity in nine rat organs using three selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine. For all substrates the highest beta-elimination rates were found in kidney, followed by liver, while brain, spleen, heart, large and small intestine, thyroid and lung were of minor importance. Since liver plays an important role in beta-elimination, hepatic beta-lyase activity was extensively studied using 23 selenocysteine Se-conjugates and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine and was compared with previously obtained renal beta-lyase data. The results showed that hepatic beta-lyase activities were 4-25-fold lower than the corresponding renal beta-lyase activities. Hepatic beta-elimination of the substrates appeared to be exclusively catalyzed by the pyridoxal 5'-phosphate-dependent beta-lyase enzyme kynureninase. Studies performed with human hepatic cytosols of three individuals showed that hepatic beta-lyase activity was 2-5-fold higher when compared with the previously obtained human renal activity. Significant correlation was obtained between human hepatic beta-lyase activities of three individuals. The relevance of this data for using SeCys-conjugates as chemopreventive and a chemoprotective agent is discussed. Based on the large differences in organ-selective beta-elimination and specific beta-lyase activity between rat and humans, the rat might not be a good model to investigate nephrotoxicity of cysteine S-conjugates, and chemoprevention and chemoprotection of SeCys-conjugates in man.     

INK4a-deficient human diploid fibroblasts are resistant to RAS-induced senescence

The CDKN2A tumour suppressor locus encodes two distinct proteins, p16(INK4a) and p14(ARF), both of which have been implicated in replicative senescence, the state of permanent growth arrest provoked in somatic cells by aberrant proliferative signals or by cumulative population doublings in culture. Here we describe primary fibroblasts from a member of a melanoma-prone family who is homozygous for an intragenic deletion in CDKN2A. Analyses of the resultant gene products imply that the cells are p16(INK4a) deficient but express physiologically relevant levels of a frameshift protein that retains the known functions of p14(ARF). Although they have a finite lifespan, the cells are resistant to arrest by oncogenic RAS. Indeed, ectopic expression of RAS and telomerase (hTERT) results in outgrowth of anchorage-independent colonies that have essentially diploid karyotypes and functional p53. We find that in human fibroblasts, ARF is not induced demonstrably by RAS, pointing to significant differences between the proliferative barriers implemented by the CDKN2A locus in different cell types or species.     

Identification of a novel HLA-B60-restricted T cell epitope of the minor histocompatibility antigen HA-1 locus

The polymorphic minor histocompatibility Ag HA-1 locus encodes two peptides, HA-1(H) and HA-1(R), with a single amino acid difference. Whereas the immunogenicity of the HA-1(R) allele has not yet been shown, the nonameric HA-1(H) peptide induces HLA-A2-restricted cytotoxic T cells in vivo and in vitro. It is not known whether the mHag HA-1(H) or HA-1(R) associates with other HLA class I molecules. Therefore, the polymorphic regions of both HA-1 alleles were analyzed to identify HLA class I binding peptides that are properly processed by proteasomal degradation. Peptide binding analyses were performed for all nonameric HA-1(H/R) peptides for binding to nine HLA class I molecules with >10% prevalence in the Caucasian population and for seven nonameric/decameric HA-1(H/R) peptides predicted to bind to HLA-A3, -B14, and -B60. Only the nonameric KECVL(H)/(R)DDL and decameric KECVL(H)/(R)DDLL peptides showed strong and stable binding to HLA-B60. In vitro digestion of 29-aa-long HA-1 peptides by purified 20S proteasomes revealed proper cleavage at the COOH termini of both HLA-B60 binding HA-1(H) and HA-1(R) peptides. In subsequent analyses, dendritic cells pulsed with the nonameric HA-1(R) peptide did not induce CTLs that recognize the natural HLA-B60/HA-1(R) ligand. In contrast, dendritic cells pulsed with the nonameric HA-1(H) peptide induced IFN-gamma-secreting T cells specific for the natural HLA-B60/HA-1(H) ligand in three HLA-B60(+) HA-1(RR) individuals, demonstrating the immunogenicity of the HLA-B60/HA-1(H) ligand. In conclusion, this study shows a novel HLA-B60-restricted T cell epitope of the minor histocompatibility Ag HA-1 locus.     

Cross-talk between hematopoiesis and angiogenesis signaling pathways

The relationship between hematopoietic cells and endothelial cells has been seen as an indication that a common progenitor, the hemangioblast, gives rise to both cell types in the yolk sac, the initial site of hematopoiesis and blood vessel formation during mammalian development. The existence of angioblast-like circulating endothelial precursor cells in adults humans has recently been suggested. In this review, we have summarized the principle mechanisms involved in the cross-talk signaling pathway between hematopoiesis and angiogenesis in order to further understand how the hematopoietic and vascular systems are established during the development.     

Detecting single molecules launched from liquid surfaces in a time-of-flight mass spectrometer using ultraviolet and infrared lasers

The launch of molecules from liquid surfaces in a time of flight mass spectrometer has been investigated using different sample preparation techniques, and by exposing the liquid samples to two different laser wavelengths, 337 nm from a N2 ultraviolet laser and 10.6 microm from a CO2 infrared laser. The molecules were detected with cryodetectors measuring the energy of the individual molecules. We present insulin and lysozyme results from samples introduced into the vacuum through a micromachined silicon injector, and from samples consisting of a glycerol droplet deposited directly on the sample holder at the high voltage stage of the ion optics.