Pre-symptomatic detection of prions by cyclic amplification of protein misfolding

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders affecting humans and animals. At present, it is not possible to recognize individuals incubating the disease before the clinical symptoms appear. We investigated the effectiveness of the "Protein Misfolding Cyclic Amplification" (PMCA) technology to detect the protease-resistance disease-associated prion protein (PrP(res)) in pre-symptomatic stages. PMCA allowed detection of PrP(res) in the brain of pre-symptomatic hamsters, enabling a clear identification of infected animals as early as two weeks after inoculation. Furthermore, PMCA was able to amplify minute quantities of PrP(res) from a variety of experimental and natural TSEs. Finally, PMCA allowed the demonstration of PrP(res) in an experimentally infected cow 32 month post-inoculation, that did not show clinical signs and was negative by standard Western blot analysis. Our findings indicate that PMCA may be useful for the development of an ultra-sensitive diagnostic test to minimize the risk of further propagation of TSEs.     

Functional and genetic analysis of two CD8 T cell subsets defined by the level of CD45RC expression in the rat

Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RC(high) CD8 T cells produce IL-2 and IFN-gamma while CD45RC(low) CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RC(high)/CD45RC(low) CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RC(high)/CD45RC(low) CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F(2)(LEW x BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RC(high)/CD45RC(low) CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RC(high)/CD45RC(low) CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.     

Multiple Mating,Paternity and Complex Fertilisation Patterns in the Chokka Squid Loligo reynaudii

Polyandry is widespread and influences patterns of sexual selection, with implications for sexual conflict over mating. Assessing sperm precedence patterns is a first step towards understanding sperm competition within a female and elucidating the roles of male- and female-controlled factors. In this study behavioural field data and genetic data were combined to investigate polyandry in the chokka squid Loligo reynaudii. Microsatellite DNA-based paternity analysis revealed multiple paternity to be the norm, with 79% of broods sired by at least two males. Genetic data also determined that the male who was guarding the female at the moment of sampling was a sire in 81% of the families tested, highlighting mate guarding as a successful male tactic with postcopulatory benefits linked to sperm deposition site giving privileged access to extruded egg strings. As females lay multiple eggs in capsules (egg strings) wherein their position is not altered during maturation it is possible to describe the spatial / temporal sequence of fertilisation / sperm precedence There were four different patterns of fertilisation found among the tested egg strings: 1) unique sire; 2) dominant sire, with one or more rare sires; 3) randomly mixed paternity (two or more sires); and 4) a distinct switch in paternity occurring along the egg string. The latter pattern cannot be explained by a random use of stored sperm, and suggests postcopulatory female sperm choice. Collectively the data indicate multiple levels of male- and female-controlled influences on sperm precedence, and highlights squid as interesting models to study the interplay between sexual and natural selection.     

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria.     

Decrease of lymphoid dendritic cells in blood from malaria-infected pregnant women

Activation of dendritic cells (DCs) during malaria is poorly documented and has mainly been studied in rodent models. We conducted studies in Senegal to better understand the relationship between DC subset activation and susceptibility of pregnant women to malaria. For each woman, samples were collected at delivery from peripheral (WB), placental (PB) and cord blood (CB). The ex vivo phenotypes of DCs were assessed using flow cytometry on whole blood. The percentage of total DCs was the same for malaria-infected or non-infected pregnant women, except for PB where a decrease in DCs was observed during infection. Lymphoid dendritic cells (LDC) also decreased in the three blood compartments of infected pregnant women and less differentiated DCs (ldDCs) increased. During infection, Human Leucocyte Antigen DR (HLA-DR) expression decreased on LDCs, myeloid DCs (MDCs) and ldDCs. IL-10 increased in the three blood compartments. These data demonstrate a modulation of DC sub-populations during placental malaria. A decrease in LDCs during placental malaria could trigger major alterations in the immune response and a change in the Th1/Th2 balance. However, elevated IL-10 observed during infection substantiates a normal micro-environment triggering normal production of DCs. The decrease in LDCs could thus be due to their migration towards spleen or other lymphoid organs.     

Immunoregulation by Vbeta specific antibodies in myasthenia gravis: mining physiological T cell homeostasis for TCR specific therapy.

Besides regulatory T cells, also comprising T cell receptor (CR)-specific T cells, it is increasingly evident that natural autoantibodies, among which anti-TCR antibodies represent additional immunomodulators in the immune system. We took advantage of myasthenia gravis (MG), a well-characterized antibody-mediated autoimmune disease, to demonstrate that without prior vaccination against TCR determinants, patients with MG present increased circulating anti-TCR antibodies directed to the dominant TCR used by pathogenic T cells. These findings, pointing to a regulatory protective role of anti-TCR antibodies, are discussed in the context of the mechanisms of action and the physiological role of anti-TCR antibodies in T cell homeostasis, and of the puzzling world of regulatory T cells. Natural anti-TCR antibodies are found in the serum of all individuals, with prevalence in physiological and pathological situations such as ageing, pregnancy, allograft transplantation, retroviral infection, and autoimmune diseases, including MG. The common link is the mounting of immune responses against alloantigens, pathogens or autoantigens, conferring on anti-TCR antibodies a broader role in controlling responses to any antigen (self or non-self) and more generally in T cell homeostasis. This homeostasis mechanism may well be exploited in therapeutic strategies based on TCR peptide vaccination in autoimmune diseases.     

Reactive oxygen species induce apoptosis of synoviocytes in vitro. Alpha-tocopherol provides no protection

Reactive oxygen species (ROS) are released during the inflammation of the synovial membrane associated with cartilage degradation in osteoarthritis. In this work, we exposed synoviocytes to superoxide anions at concentrations that may cause either apoptosis or necrosis. We studied membrane organization, dehydrogenase mitochondrial activity and nuclear morphology and integrity, to determine the nature of the death process initiated by superoxide anions and tried to counteract ROS effects with alpha-tocopherol. We found that oxidative stress caused synoviocytes to undergo a process of cell death of an apoptotic nature rather than necrotic. Mitochondrial injury occurred at an early stage, and the FITC-annexin-V-positive/propidium iodide-positive cells occurred later than the metabolic changes. DNA strand breaks were evident at 8 h and nuclear condensation at 24 h. No LDH activity was detected in culture supernatants. In our experimental conditions, alpha-tocopherol had little effect on stress damage; the antioxidant properties of this molecule did not affect the apoptosis caused by superoxide anions.     

Cell adhesion and integrin expression are modulated by oxidative stress in EA.hy 926 cells

The effects of oxidative stress on integrin-mediated cell adhesion to the extracellular matrix (ECM) and related apoptosis were investigated using the EA.hy926 endothelial cells treated (or not) with two oxidants: the hypoxanthine/xanthine oxidase system (HX/XO) or the tert-butyl hydroperoxide (t-BHP) which both increased cell apoptosis. Cell adhesion onto vitronectin (Vn) and fibronectin (Fn) was increased at low concentrations of HX/XO (up to 5 mU/ml) or t-BHP (up to 125 microM) and prevented ROS-induced apoptosis. Flow cytometry analysis of integrin expression showed that the expression of integrin alphav and alpha5 subunits was, respectively, increased and decreased. Cell adhesion inhibition experiments using function-blocking monoclonal antibodies against integrin subunits indicated that alphavbeta1 and alphavbeta3 integrins were involved in adhesion of cells to Vn, and alphavbeta3 integrin played a major role in oxidant-treated cells. For adhesion to Fn, alpha5beta1 and alphavbeta1 integrins were required for oxidant-treated cells. Taken together, the results suggest that reactive oxygen species (ROS) produced either by HX/XO or t-BHP could affect expression and/or activation of specific integrins in the interaction of EA.hy926 cells with ECM.