Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor–mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins

Placenta-Like Structure of the Aphid Endoparasitic Wasp Aphidius ervi: A Strategy of Optimal Resources Acquisition

Aphidius ervi (Hymenoptera: Braconidae) is an entomophagous parasitoid known to be an effective parasitoid of several aphid species of economic importance. A reduction of its production cost during mass rearing for inundative release is needed to improve its use in biological control of pests. In these contexts, a careful analysis of its entire development phases within its host is needed. This paper shows that this parasitoid has some characteristics in its embryological development rather complex and different from most other reported insects, which can be phylogenetically very close. First, its yolkless egg allows a high fecundity of the female but force them to hatch from the egg shell rapidly to the host hemocoel. An early cellularisation allowing a rapid differentiation of a serosa membrane seems to confirm this hypothesis. The serosa wraps the developing embryo until the first instar larva stage and invades the host tissues by microvilli projections and form a placenta like structure able to divert host resources and allowing nutrition and respiration of embryo. Such interspecific invasion, at the cellular level, recalls mammal's trophoblasts that anchors maternal uterine wall and underlines the high adaptation of A. ervi to develop in the host body

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T-cells

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor-mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins

Identification of 12 new susceptibility loci for different histotypes of epithelial ovarian cancer.

To identify common alleles associated with different histotypes of epithelial ovarian cancer (EOC), we pooled data from multiple genome-wide genotyping projects totaling 25,509 EOC cases and 40,941 controls. We identified nine new susceptibility loci for different EOC histotypes: six for serous EOC histotypes (3q28, 4q32.3, 8q21.11, 10q24.33, 18q11.2 and 22q12.1), two for mucinous EOC (3q22.3 and 9q31.1) and one for endometrioid EOC (5q12.3). We then performed meta-analysis on the results for high-grade serous ovarian cancer with the results from analysis of 31,448 BRCA1 and BRCA2 mutation carriers, including 3,887 mutation carriers with EOC. This identified three additional susceptibility loci at 2q13, 8q24.1 and 12q24.31. Integrated analyses of genes and regulatory biofeatures at each locus predicted candidate susceptibility genes, including OBFC1, a new candidate susceptibility gene for low-grade and borderline serous EOC

Immobilization of growth factors using cellulose binding domain-cytokine fusion proteins

This study describes the application and characterization of cellulose binding domain (CBD)- cytokine fusion proteins for the cultivation of cytokine dependent cells. The affinity, binding reversibility and surface diffusivity of type-U CBDs are analyzed. Binding of CBDs to crystalline cellulose is irreversible with respect to protein dilution in the solution phase. The surface diffusion rates of an exo-β-l-4-glycanase (Cex), an endo- β -l-4-glucanase (CenA), and their respective isolated CBDs, are quantified using fluorescence recovery after photobleaching analysis. Greater than 70% of bound molecules are mobile on the cellulose surface (Dsurf ~ 3 x 10 ⁻¹¹ cm² /sec). Surface diffusion rates are dependent on surface coverage density and temperature ( Ea ~ 45 kJ/mol.K). These attributes of the C B D suggest that C B D fusion proteins are not simply immobilized on the cellulose surface, but rather localized at it, so as to retain 2- dimensional mobility. Localization of cytokines to cellulose via a C B D affinity tag, provides a convenient method for adsorbing growth factors to a solid phase (cellulose) in a surface-active form. Bioactivity and long term stability of CBD-cytokine adsorption to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound fusion proteins of murine stem cell factor with CBDcex (CBD-SCF), murine interleukin-3 with CBDCenA (CBD-IL3) and murine interleukin-2 with CBDcenA (CBD-IL2), stimulate the proliferation of their respective factor dependent cells. Cellulose localization of CBD-SCF results in a significant increase in the persistence of tyrosine activation of the SCF receptor. Furthermore, when cells are incubated with cellulose-bound CBD-SCF, activated receptors and CBD-SCF co-localize at the cellulose surface. In contrast to the surface diffusion of CBDC ex, CBD-SCF does not diffuse on crystalline cellulose. This is likely a result of the formation of CBD-SCF dimers (through SCF domain dimerization) at the cellulose interface.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat

Maximizing the expression of a cloned gene under the regulation of the metallothionein promoter

A stably transformed BHK cell line, engineered to produce a human transferrin half-molecule under the control of a mouse metallothionein promoter, was used as a model system to develop strategies to maximize recombinant protein productivity in mammalian cell culture. The metallothionein promoter is inducible and promotes high levels of expression. Fully induced cells produced up to 0.7 pg transferrin/cell hr, a 4-fold increase in transferrin production over uninduced levels. Cell growth was inhibited at cadmium dosages above 1.0 fmole/cell; prolonged exposure at this dosage is cytotoxic. The metal dose-dependence of induction and lethal effects at high metal dosages motivated the development of special strategies to maximize gene expression. Dosing regimes which maintained cell associated cadmium concentrations below 0.25 fmole/cell, ensured cell growth and high cell specific productivities which maximized final product titers. For routine batch culture initial inducer loadings of 10 fmole/cell for zinc and 1.0 fmole/cell for cadmium gave near-maximum transferrin production levels. For extended culture, repeated small doses of between 0.25 fmole/cell and 0.45 fmole/cell, based on initial cell counts, every 48 hours maximized transferrin synthesis with this cell line. A novel compartmental model with 3 inducer pools was developed which quantitatively reproduced many of the experimental results. Dose-dependent compartmental transfer functions were modeled and kinetic parameters estimated for transferrin synthesis and secretion. Several batch and fedbatch inducer dosing strategies were simulated to identify protocols which maximized gene expression. Sensitivity analysis of the model's parameters predicted that transferrin production rates are less sensitive to variation in cell specific cadmium uptake rates at lower inducer loadings.Applied Science, Faculty ofChemical and Biological Engineering, Department ofGraduat