Cutting edge: soluble IL-6R is produced by IL-6R ectodomain shedding in activated CD4 T cells

IL-6 trans-signaling via the soluble IL-6R (sIL-6R) plays an important role in the progression of several autoimmune diseases and cancer by providing IL-6-responsiveness to cells lacking IL-6R. However, the potential sources of sIL-6R are less understood. In this study we show that sIL-6R is produced by both naive and memory CD4 T cells upon TCR activation. The production of sIL-6R by activated CD4 T cells is mediated by shedding of the membrane-bound IL-6R, and this process correlates with the expression of the metalloproteinase ADAM17 in these cells. In contrast to CD4 T cells, CD8 T cells do not express ADAM17 and their production of sIL-6R is negligible. Thus, during an immune response CD4 T cells are an important source of sIL-6R. Production of sIL-6R by autoreactive CD4 T cells may contribute to their role in the development of autoimmune disease by conferring IL-6-responsiveness to cells lacking IL-6R such as synoviocytes.     

Life's unity and flexibility: the ecological link.

The small size, ubiquity, metabolic versatility and flexibility, and genetic plasticity (horizontal transfer) of microbes allow them to tolerate and quickly adapt to unfavorable and/or changing environmental conditions. Prokaryotes are endowed with sophisticated cellular envelopes that contain molecules not found elsewhere in the biological world. Although prokaryotic cells lack the organelles that characterize their eukaryotic counterparts, their interiors are surprisingly complex. Prokaryotes sense their environment and respond as individual cells to specific environmental challenges; but prokaryotes also act cooperatively, displaying communal activities. In many microbial ecosystems, the functionally active unit is not a single species or population (clonal descendence of the same bacterium) but a consortium of two or more types of cells living in close symbiotic association. Only recently have we become aware that microbes are the basis for the functioning of the biosphere. Thus, we are at a unique time in the history of science, in which the interaction of technological advances and the exponential growth in our knowledge of the present microbial diversity will lead to significant advances not only in microbiology but also in biology and other sciences in general.     

The sodium channel of human excitable cells is a target for gambierol.

BACKGROUND: Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized. METHODS: We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium. RESULTS: We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells. CONCLUSION: These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.     

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.     

E- and P-selectin are not required for the development of experimental autoimmune encephalomyelitis in C57BL/6 and SJL mice

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier (BBB) and gain access to the CNS. It is well-established that alpha4 integrins are actively involved in leukocyte recruitment across the BBB during EAE. In contrast, the role of endothelial E- and P-selectin in this process has been a controversial issue. In this study, we demonstrate that P-selectin protein can be detected in meningeal blood vessel endothelial cells in healthy SJL and C57BL/6 mice and on rare parenchymal CNS blood vessels in C57BL/6, but not SJL, mice. During EAE, expression of P-selectin but not E-selectin was found up-regulated on inflamed CNS microvessels surrounded by inflammatory infiltrates irrespective of their meningeal or parenchymal localization with a more prominent immunostaining detected in C57BL/6 as compared with SJL mice. P-selectin immunostaining could be localized to CNS endothelial cells and to CD41-positive platelets adhering to the vessel wall. Despite the presence of P-selectin in wild-type mice, E/P-selectin-deficient SJL and C57BL/6 mice developed clinical EAE indistinguishable from wild-type mice. Absence of E- and P-selectin did neither influence the activation of myelin-specific T cells nor the composition of the cellular infiltrates in the CNS during EAE. Finally, endothelial-specific tetracycline-inducible expression of E-selectin at the BBB in transgenic C57BL/6 mice did not alter the development of EAE. Thus, E- and P-selectin are not required for leukocyte recruitment across the BBB and the development of EAE in C57BL/6 and in SJL mice.     

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.     

The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis

This study reports a new capture ELISA (MM3-SERO) for the serodiagnosis of sheep fascioliasis, based on the use of the monoclonal antibody (mAb) MM3. Like our previously reported indirect ELISA method, based on the use of a FPLC-purified fraction (fraction IV) of the Fasciola hepatica excretion/secretion antigens (ESAs), this new test was able to detect animals infected with very small numbers of metacercariae (5-40) and showed no cross-reaction with sera from sheep infected with other parasites, i.e., Moniezia spp., Cysticercus tenuicollis, and Dicrocoelium dendriticum. In contrast with these 2 methods, some sera (mainly those obtained from animals infected with D. dendriticum) showed high reactivities in indirect ELISA with whole F. hepatica ESAs used as control. Interestingly, the MM3-SERO ELISA has a better signal-to-noise ratio than the fraction-IV ELISA, thus allowing detection of seroconversion in infected sheep on average 1 wk earlier (3.2 +/- 0.4 wk postinfection [PI] for MM3-SERO ELISA vs. 4.2 +/- 0.9 wk PI for fraction IV ELISA). Moreover, the antibody response detected with MM3-SERO ELISA was more uniform, with seroconversion always occurring at 4 wk PI in sheep with 1-2 flukes and at 3 wk PI in sheep with more than 2 flukes. The MM3-SERO ELISA was also used to evaluate the kinetics of antibody response against MM3-recognized antigens in sera from sheep experimentally infected with F. hepatica and then treated with triclabendazole. Our results showed that antibody levels dropped by about 25% during the 4-wk observation period following the flukicide treatment, whereas they remained invariably high in all sheep left untreated. We conclude that the MM3-SERO ELISA is a 100% sensitive and 100% specific test for the early serodiagnosis of sheep fascioliasis. Preliminary studies in our laboratory seem to indicate that this method may also be useful for the determination of anti-F. hepatica antibodies in serum and milk of other ruminants. A commercial version of MM3-SERO is currently available from BIO X Diagnostics (La Jemelle, Belgium).     

DNA polymerase beta catalytic efficiency mirrors the Asn279-dCTP H-bonding strength

Ternary complexes of wild type or mutant form of human DNA polymerase beta (pol beta) bound to DNA and dCTP substrates were studied by molecular dynamics (MD) simulations. The occurrences of contact configurations (CC) of structurally important atom pairs were sampled along the MD trajectories, and converted into free-energy differences, DeltaG(CC). DeltaG(CC) values were correlated with the experimental binding and catalytic free energies for the wild type pol beta and its Arg183Ala, Tyr271Ala, Asp276Val, Lys280Gly, Arg283Ala, and Glu295Ala mutants. The correlation coefficients show that the strength of the H-bond between dCTP and Asn279 is a strong predictor of the mutation-induced changes in the catalytic efficiency of pol beta. This finding is consistent with the view that enzyme preorganization plays a major role in controlling DNA polymerase specific activity.     

Carboxy terminal extended phytocystatins are bifunctional inhibitors of papain and legumain cysteine proteinases

Plant legumains are cysteine proteinases putatively involved in processing endogenous proteins. Phytocystatins (PhyCys) have been described as plant inhibitors of papain-like cysteine proteinases. Some PhyCys contain a carboxy terminal extension with an amino acid motif (SNSL) similar to that involved in the inhibition of legumain-like proteins by human cystatins. The role of these carboxy terminal extended PhyCys as inhibitors of legumain-like cysteine proteinases is here shown by in vitro inhibition of human legumain and legumain-like activities from barley extracts. Moreover, site-directed mutagenesis has demonstrated that the asparagine of the SNSL motif is essential in this inhibition. We prove for first time the existence of legumain inhibitors in plants.     

Quantitative trait loci controlling cyanogenic glucoside and dry matter content in cassava (Manihot esculenta Crantz) roots

Cassava (Manihot esculenta Crantz) is a starchy root crop grown in the tropics mainly by small-scale farmers even though agro-industrial processing is rapidly increasing. For this processing market improved varieties with high dry matter root content (DMC) is required. Potentially toxic cyanogenic glucosides are synthesized in the leaves and translocated to the roots. Selection for varieties with low cyanogenic glucoside potential (CNP) and high DMC is among the principal objectives in cassava breeding programs. However, these traits are highly influenced by the environmental conditions and the genetic control of these traits is not well understood. An S(1) population derived from a cross between two bred cassava varieties (MCOL 1684 and Rayong 1) that differ in CNP and DMC was used to study the heritability and genetic basis of these traits. A broad-sense heritability of 0.43 and 0.42 was found for CNP and DMC, respectively. The moderate heritabilities for DMC and CNP indicate that the phenotypic variation of these traits is explained by a genetic component. We found two quantitative trait loci (QTL) on two different linkage groups controlling CNP and six QTL on four different linkage groups controlling DMC. One QTL for CNP and one QTL for DMC mapped near each other, suggesting pleiotrophy and/or linkage of QTL. The two QTL for CNP showed additive effects while the six QTL for DMC showed additive effect, dominance or overdominance. This study is a first step towards developing molecular marker tools for efficient breeding of CNP and DMC in cassava.