Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors.

TNF receptor family members fused to the constant domain of immunoglobulin G have been widely used as immunoadhesins in basic in vitro and in vivo research and in some clinical applications. In this study, we assemble soluble, high avidity chimeric receptors on a pentameric scaffold derived from the coiled-coil domain of cartilage oligomeric matrix protein (COMP). The affinity of Fas and CD40 (but not TNFR-1 and TRAIL-R2) to their ligands is increased by fusion to COMP, when compared to the respective Fc chimeras. In functional assays, Fas:COMP was at least 20-fold more active than Fas:Fc at inhibiting the action of sFasL, and CD40:COMP could block CD40L-mediated proliferation of B cells, whereas CD40:Fc could not. In conclusion, members of the TNF receptor family can display high specificity and excellent avidity for their ligands if they are adequately multimerized.

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.

Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Membrane proteins are notoriously difficult to express in a soluble form. Here, we use wheat germ cell-free expression in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV). We show that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free reaction. This finding can be explained by the low critical micelle concentration (CMC) of these detergents, which keeps the monomer concentrations low while at the same time providing the necessary excess of detergent concentration above CMC required for full target protein solubilization. We estimate that a tenfold excess of detergent micelles with respect to the protein concentration is sufficient for solubilization, a number that we propose as a guideline for detergent screening assays.

A Novel HLA-B18 Restricted CD8+ T Cell Epitope Is Efficiently Cross-Presented by Dendritic Cells from Soluble Tumor Antigen.

NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+) T cell epitope, NY-ESO-1(88-96) (LEFYLAMPF) and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165) epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96) is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165). On the other hand, NY-ESO-1(157-165) is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35); whereas NY-ESO-1(88-96) was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96) is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+) melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96) from patients, including those who received NY-ESO-1 ISCOMATRIX? vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+) T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

Characterisation of two soluble metalloexopeptidases in the protozoan parasite Leishmania major.

Two soluble exopeptidases were identified in promastigotes of Leishmania major, using an iodinated model tetrapeptide (LIAY) as substrate. Similar activities were also detected in L. major amastigotes and in different species of Leishmania promastigotes. A carboxy- and an aminopeptidase activity were resolved and isolated by anion exchange and gel permeation chromatographies. A single polypeptide of 62 kDa co-purified with the aminopeptidase activity. Optimum pH was neutral for the carboxypeptidase and neutral to alkaline for the aminopeptidase. Both activities were able to hydrolyse a dipeptide substrate (YL), and were inhibited by 20 microM bestatin and 200 microM 1,10-phenanthroline, but not by leupeptin, iodoacetamide and a range of other inhibitors. These results strongly suggest that both enzymes are metalloexopeptidases and thus represent a novel class of soluble peptidases in Leishmania.

Integrin-mediated adhesion and soluble ligand binding stabilize COX-2 protein levels in endothelial cells by inducing expression and preventing degradation.

Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin alphaVbeta3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041-1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838-45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-L-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-L-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways.

Thermoplastic Starch-based Composites Reinforced with Rape Fibers: Water Uptake and Thermomechanical Properties

Fully biodegradable composite materials were obtained through reinforcement of a commercially available thermoplastic starch (TPS) matrix with rapeseed fibers (RSF). The influence of reinforcement content on the water sorption capacity, as well as thermal and thermo-mechanical properties of composites were evaluated. Even though the hydrophilic character of natural fibers tends to favor the absorption of water, results demonstrated that the incorporation of RSF did not have a significant effect on the water uptake of the composites. DSC experiments showed that fibers restricted the mobility of the starch macromolecules from the TPS matrix, hence reducing their capacity to crystallize. The viscoelastic behaviour of TPS was also affected, and reinforced materials presented lower viscous deformation and recovery capacity. In addition, the elasticity of materials was considerably diminished when increasing fiber content, as evidenced in the TMA and DMTA measurements

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Local ocular immunomodulation resulting from electrotransfer of plasmid encoding soluble TNF receptors in the ciliary muscle.

PURPOSE: Plasmid electrotransfer in the ciliary muscle allows the sustained release of therapeutic proteins within the eye. The aim of this study was to evaluate whether the ocular production of TNF-alpha soluble receptor, using this nonviral gene therapy method, could have a beneficial local effect in a model of experimental autoimmune uveoretinitis (EAU). METHODS: Injection of a plasmid encoding a TNF-alpha p55 receptor (30 microg) in the ciliary muscle, combined with electrotransfer (200 V/cm), was carried out in Lewis rat eyes 4 days before the induction of EAU by S-antigen. Control eyes received naked plasmid electrotransfer or simple injection of the therapeutic plasmid. The disease was evaluated clinically and histologically. Cytokines and chemokines were analyzed in the ocular media by multiplex assay performed 15 and 21 days after immunization. RESULTS: Ocular TNF-alpha blockade, resulting from the local secretion of soluble receptors, was associated with delayed and significantly less severe uveitis, together with a reduction of the retinal damages. Compared with the controls, treated eyes showed significantly lower levels of IL-1beta and MCP1, higher levels of IL-13 and IL-4, and reduced NOS-2 expression in infiltrating cells. Treatment did not influence TNF-alpha levels in inguinal lymph nodes. CONCLUSIONS: Taken together, these results indicate that local immunomodulation was achieved and that no systemic adverse effects of TNF-alpha blockade observed after systemic injection of TNF-alpha inhibitors should be expected.

Availability of a soluble phosphorus source applied to soil samples with different acidicity levels

Considerations on the interactions of P in the soil-plant system have a long history, but are still topical and not yet satisfactorily understood. One concern is the effect of liming before or after application of soluble sources on the crop yield and efficiency of available P under these conditions. The aim of this study was to evaluate the effect of soil acidity on availability of P from a soluble source, based on plant growth and chemical extractants. Nine soil samples were incubated with a dose of 200 mg kg-1 P in soil with different levels of previously adjusted acidity (pH H2O 4.5; 5.0; 5.5; 6.0 and 6.5) and compared to soils without P application. After 40 days of soil incubation with a P source, each treatment was limed again so that all pH values were adjusted to 6.5 and then sorghum was planted. After the first and second liming the P levels were determined by the extractants Mehlich-1, Bray-1 and Resin, and the fractionated inorganic P forms. In general, the different acidity levels did not influence the P availability measured by plant growth and P uptake at the studied P dose. For some soils however these values increased or decreased according to the initial soil pH (from 4.5 to 6.5). Plant growth, P uptake and P extractable by Mehlich-1 and Bray-1 were significantly correlated, unlike resin-extractable P, at pH values raised to 6.5. These latter correlations were however significant before the second liming. The P contents extracted by Mehlich-1 and Bray-1 were significantly correlated with each other in the entire test range of soil acidity, even after adjusting pH to 6.5, besides depending on the soil buffering capacity for P. Resin was also sensitive to the properties that express the soil buffering capacity for P, but less clearly than Mehlich-1 and Bray-1. The application of triple superphosphate tended to increase the levels of P-Al, P-Fe and P-Ca and the highest P levels extracted by Bray-1 were due to a higher occurrence of P-Al and P-Fe in the soils.

Water-soluble nutrients in aerial plant parts of peanut and white oat as affected by lime and gypsum application

Lime and gypsum influence nutrient availability and uptake, as well as the content of organic acids in the aerial plant parts. These changes, quantified by plant analysis of soluble nutrients, may potentiate the effect of soil amendment, ensuring the sustainability of the no-tillage system. In this sense the effect of lime and gypsum surface application on the content of water-soluble nutrients in peanut and oat residues was evaluated. The experiment was conducted on an Oxisol in Botucatu (SP) in the growing seasons 2004/2005 and 2005/2006. It was arranged in a randomized block design in split plots with four replications, where lime rates represented the plots and presence or absence of gypsum application the subplots. Peanut was grown in summer and white oat in the winter in the entire experimental area. Gypsum applied to peanut increased soluble Ca only in the first season, due to the short period between product application and determination of soluble nutrient contents in the plant extract. Liming of peanut and oat increased soluble Ca, Mg, K contents, did not alter Cu content and reduced Zn, Mn and Fe contents in both years of cultivation. Gypsum on the other hand reduced the electrical conductivity of peanut (2004/2005 and 2005/2006) and white oat (2004/2005).

Arbuscular mycorrhizal inoculation increases biomass of Euterpe edulis and Archontophoenix alexandrae after two years under field conditions

Inoculation with arbuscular mycorrhizal fungi (AMF) of tree seedlings in the nursery is a biotechnological strategy to improve growth, survival after transplanting, biomass production and to reduce the use of fertilizers. Archontophoenix alexandrae and Euterpe edulis are palm species used in southern Brazil to produce the palm heart, the latter being included in the list of threatened species due to the overexploitation of its native population. The purpose of this paper was to evaluate the effect of mycorrhizal inoculation on growth and physiological parameters of A. alexandrae and E. edulis. After germination, the seedlings were inoculated (AMF) or not (CTL) with AMF in the treatments. Values of chlorophyll content, biomass and shoot phosphorus were not statistically different between the AMF and CTL treatments, after five months in the greenhouse. Inoculation with AMF significantly increased the levels of starch and soluble carbohydrates in shoots and roots of both species. Under field conditions, AMF had no effect on stem diameter and height after 12 and 24 months, but total plant biomass and leaf, stem and root biomass were greater in AMF than in CTL plants. The data indicated that AMF inoculation in the nursery has a strong effect on biomass accumulation after growing for 24 months under field conditions. Therefore, AMF inoculation should be considered an important strategy to increase growth and production of these economically important tropical palm species.

Molecular weight of hydroxyethyl starch : is there an effect on blood coagulation and pharmacokinetics ?

Résumé Fondement : le développement de solutions d'hydroxy-éthyl-amidons (HEAS) avec peu d'impact sur la coagulation sanguine, mais un effet supérieur sur la volémie, par comparaison aux HEAS couramment utilisés, est d'un grand intérêt clinique. Nous posons l'hypothèse que des solutions de haut poids moléculaire et de bas degré de substitution possèdent ces caractéristiques. Méthode : trente porcs ont été perfusés avec trois HEAS différents (20 ml/kg) de même degré de substitution (0.42) mais de poids moléculaire différent (130, 500 et 900 kDa). Une série de prélèvements sanguins ont été effectués sur 24 heures, sur lesquels des analyses de coagulation sanguine étaient effectuées par thromboélastographie et dosages plasmatiques. De plus, la concentration plasmatique ainsi que le poids moléculaire in vivo ont été déterminés, ainsi que des paramètres de pharmacocinétiques, ceci en se basant sur un modèle bi-compartimental. Résultats : les analyses de thromboélastographie et les tests de coagulation plasmatique n'ont pas démontré d'altération plus marquée de la coagulation sanguine après l'utilisation des solutions des HAES 500 et HAES 900, par comparaison avec celle de HAES 130. Par contre, les HAES 500 et HAES 900 ont présenté une plus grande aire sous la courbe (area under the curve), dans la relation concentration en fonction du temps [1542 (142) g min litre-1, p<0.001, 1701 (321) g min litre-1, p<0.001] par rapport au HAES 130 [1156 (223) g min litre-1]. La demi-vie alpha (t ½α) était plus longue pour les HAES 500 [53.8 (8.6) min, p<0.01] et HAES 900 [57.1 (12.3) min, p<0.01 ]que pour le HAES 130 [39.9 (10.7) min]. La demi-vie beta (t½β) était par contre similaire pour les trois types de HAES [de 332 (100) à 381 (63) min]. Conclusions : pour les HAES de bas degré de substitution, le poids moléculaire n'est pas un facteur clé en ce qui concerne l'altération de la coagulation. La persistance intravasculaire initialement plus longue des HAES de haut poids moléculaire et bas degré de substitution pourrait résulter dans un plus long effet volémique de ces substances. Abstract Background: The development of hydroxyethyl starches (HES) with low impact on blood coagulation but higher volume effect compared with the currently used HES solutions is of clinical interest. We hypothesized that high molecular weight, low-substituted HES might possess these properties. Methods: Thirty pigs were infused with three different HES solutions (20 ml kg-1) with the same degree of molar substitution (0.42) but different molecular weights (130, 500 and 900 kDa). Serial blood samples were taken over 24 h and blood coagulation was assessed by Thromboelastograph® analysis and analysis of plasma coagulation. In addition, plasma concentration and in vivo molecular weight were determined and pharmacokinetic data were computed based on a two-compartment model. Results: Thromboelastograph analysis and plasma coagulation tests did not reveal a more pronounced alteration of blood coagulation with HES 500 and HES 900 compared with HES 130. In contrast, HES 500 and HES 900 had a greater area under the plasma concentration-time curve [1542 (142) g min litre-1, P<0.001, 1701 (321) g min litre-1, P<0.001] than HES 130 [I 156 (223) g min litre-1] and alpha half life (t ½α) was longer for HES 500 [53.8 (8.6) min, P<0.01 ] and HES 900 [57. I (I 2.3) min, P<0.01 ] than for HES 130 [39.9 (I 0.7) min]. Beta half life (t½β), however, was similar for all three types of HES [from 332 (100) to 381 (63) min]. Conclusions. In low-substituted HES, molecular weight is not a key factor in compromising blood coagulation. The longer initial intravascular persistence of high molecular weight lowsubstituted HES might result in a longer lasting volume effect.

Soluble carbon in oxisol under the effect of organic residue rates

The application of organic residues to the soil can increase soluble organic carbon (SOC) and affect the pH and electrolytic conductivity (EC) of the soil. However, the magnitude of these changes depends on the type of residue and the applied dose. This study aimed to evaluate the effect of increasing C rates contained in organic residue on the pH, EC, water-extractable total carbon (WETC), water-extractable organic carbon (WEOC), and water-extractable inorganic carbon (WEIC) in soil treated with manure (chicken, swine, and quail), sawdust, coffee husk, and sewage sludge. The levels of total C (TC- KH2PO4), organic carbon (OC- KH2PO4), and inorganic C (IC- KH2PO4) extractable by a 0.1 mol L-1 KH2PO4 solution were also quantified in soil under the effect of increasing rates of chicken and quail manures. The following rates of organic residue C were applied to a dystrophic Red Latosol (Oxisol) sample: 0, 2,000, 5,000, 10,000, and 20,000 mg kg-1. The addition of organic residues to the soil increased pH, except in the case of sewage sludge, which acidified the soil. The acidity correction potential of chicken and quail manure was highest, dependent on the manure rate applied; regardless of the dose used, sawdust barely alters the soil pH. At all tested rates, the EC of the soil treated with swine manure, coffee husk, and sawdust remained below 2.0 dS m-1, which is a critical level for salinity-sensitive crops. However, the application of chicken or quail manure and sewage sludge at certain rates increased the EC to values above this threshold level. Highest levels of WETC, WEOC, and WEIC were obtained when chicken and quail manure and coffee husk were applied to the Oxisol. The quantities of SOC extracted by KH2PO4 were higher than the quantities extracted by water, demonstrating the ability of soil to adsorb C into its colloids.