Increased CD8+ T cell responses to apoptotic T cell-associated antigens in multiple sclerosis.

BACKGROUND: Here, we evaluated the hypothesis that CD8(+) T cell responses to caspase-cleaved antigens derived from effector T cells undergoing apoptosis, may contribute to multiple sclerosis (MS) immunopathology. METHODS: The percentage of autoreactive CD8(+) T effector cells specific for various apoptotic T cell-associated self-epitopes (apoptotic epitopes) were detected in the peripheral blood and cerebrospinal fluid (CSF) by both enzyme-linked immunospot and dextramers of class I molecules complexed with relevant apoptotic epitopes. Moreover, the capacity of dextramer(+) CD8(+) T cells to produce interferon (IFN)-γ and/or interleukin (IL)-17 in response to the relevant apoptotic epitopes was evaluated by the intracellular cytokine staining. Cross-presentation assay of apoptotic T cells by dendritic cells was also evaluated ex vivo. RESULTS: We found that polyfunctional (IFN-γ and/or IL-17 producing) autoreactive CD8(+) T cells specific for apoptotic epitopes were represented in MS patients with frequencies significantly higher than in healthy donors. These autoreactive CD8(+) T cells with a strong potential to produce IFN-γ or IL-17 in response to the relevant apoptotic epitopes were significantly accumulated in the CSF from the same patients. In addition, the frequencies of these autoreactive CD8(+) T cells correlated with the disease disability. Cross-presentation assay revealed that caspase-cleaved cellular proteins are required to activate apoptotic epitope-specific CD8(+) T cells ex vivo. CONCLUSION: Taken together, these data indicate that apoptotic epitope-specific CD8(+) T cells with strong inflammatory potential are recruited at the level of the inflammatory site, where they may be involved in MS immunopathology through the production of high levels of inflammatory cytokines

Polyfunctional Type-1, -2, and -17 CD8+ T Cell Responses to Apoptotic Self-Antigens Correlate with the Chronic Evolution of Hepatitis C Virus Infection

Caspase-dependent cleavage of antigens associated with apoptotic cells plays a prominent role in the generation of CD8+ T cell responses in various infectious diseases. We found that the emergence of a large population of autoreactive CD8+ T effector cells specific for apoptotic T cell-associated self-epitopes exceeds the antiviral responses in patients with acute hepatitis C virus infection. Importantly, they endow mixed polyfunctional type-1, type-2 and type-17 responses and correlate with the chronic progression of infection. This evolution is related to the selection of autoreactive CD8+ T cells with higher T cell receptor avidity, whereas those with lower avidity undergo prompt contraction in patients who clear infection. These findings demonstrate a previously undescribed strict link between the emergence of high frequencies of mixed autoreactive CD8+ T cells producing a broad array of cytokines (IFN-γ, IL-17, IL-4, IL-2…) and the progression toward chronic disease in a human model of acute infection

Complex evolution in genetic networks

A simple model of diffusively coupled genetic networks on a 1d lattice is considered as a case study for characterizing complex unpredictable dynamics in Lyapunov-stable spatially extended systems. This dynamical regime, corresponding to transient evolution lasting over times that grow exponentially with the system size, is shown to be associated to the existence of a multifractal attractor in the thermodynamic limit. Space-time averages of proper observables exhibit robust statistical properties for finite, sufficiently large samples

Symbolic and Global Dynamics in Coupled Genetic Networks: Evolution of Artificial Cell Populations At the Edge of and Within Chaotic Transient Behaviour

We investigate the main dynamical features of a Coupled Map Lattice that mimics the evolution in time of genetic networks in regular cellular clusters. Each cell is represented by the same network of genes whose protein products determine mutual interactions both in space and time. The global dynamics -- quantified by the mean concentrations of the products on the overall lattice --, and the symbolic dynamics -- activity-inactivity patterns of the genes --, have been analyzed in the longtransient regime, shown by the system in certain regions of the parameter space. Although the system is linearly stable -- the maximum Lyapunov exponent is proved to be always negative --, the long transient regime exhibits stationary features proper of chaotic evolution. Moreover, in the other regions of the parameter space the dynamics shows shorter transients and more regular behaviour. The analysis of the global and symbolic dynamics allows the interpretation of these different regimes in biological..

Toward a functional analysis of salivary proteins from the African malaria vector Anopheles gambiae: a work in progress.

The mosquito salivary glands secrete a large number of compounds with anti-haemostatic, anti-inflammatory and immuno-modulatory activity that are of high adaptive value in relationship to haematophagy. Using a selective cloning strategy we have identified, in the last few years, more than twenty genes whose expression is either tissue-specific or highly enriched in the Anopheles gambiae adult female salivary glands (Arcà B et al, 1999 Proc Natl Acad Sci USA, 96: 1516-1521; Lanfrancotti A et al, 2002 FEBS Letters, 517: 67-71). Surprisingly, almost half of the genes identified so far either share only weak similarity to known proteins in the databases or do not show similarity at all; they represent therefore “orphan” proteins in search for a function. For these reasons we decided to proceed toward a functional analysis and, as a preliminary step in this direction, we started to express some of these proteins in vitro in a recombinant form. We have initially focused our attention on two small putative proteins sharing low level of similarity with anticoagulants from distantly related species: (a) gSG6 shows 24% identity and 65% similarity to AcAP6, an anti-coagulant from the haematophagous nematode Ancylostoma caninum that contains a TIL domain (Trypsin-Inhibitor-Like cystein-rich domain), typical of serine protease inhibitors; (b) gSG7 shares 19% identity and 45% similarity with a secreted phospholipase A2 from the venom of the cobra Naja naja atra that also possesses anticoagulant activity. The expression system based on the yeast Pichia pastoris was chosen because it seemed to combine advantages of prokaryotic and eukaryotic systems. The cDNAs encoding gSG6 and gSG7 were cloned downstream of the methanol-inducible alcohol oxidase promoter (AOX1); moreover, a c-myc epitope and a six-histidine tag were included to facilitate detection and purification of the recombinant proteins. The initial trials to express these two recombinant proteins in small scale shake-flask cultures were unsuccessful and, in the attempt to enhance the production of these proteins in P. pastoris, we performed high-cell density cultures in a 2 liters benchtop fermentor. Using this approach we could produce and purify by affinity chromatography on Ni-columns small amounts of the two proteins that could be used for a few functional assays. Classical Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) tests were performed to verify the ability of the recombinant proteins to affect either the extrinsic or the intrinsic pathway of the coagulation cascade. However, neither PT nor APTT showed any anti-coagulation property of the recombinant gSG6 or gSG7. Since salivary secretions of haematophagous arthropods are known to contain also immuno-modulators we are presently testing the ability of these recombinant proteins to affect the immune system assaying their effect on differentiation and maturation of dendritic cells. Preliminary results seem compatible with immune-suppressive properties of the two proteins

Toward a functional analysis of salivary proteins from the malaria vector Anopheles gambiae: a work in progress.

The mosquito salivary glands secrete a large number of compounds with anti-haemostatic, anti-inflammatory and immuno-modulatory activity that are of high adaptive value in relationship to haematophagy. Using a selective cloning strategy we previously identified twenty-two genes whose expression is either tissue-specific or highly enriched in the Anopheles gambiae salivary glands. Surprisingly, many of them do not show significant similarity to known proteins and, therefore, appear to represent “orphan” proteins in search for a function. For these reasons, as a preliminary step toward functional analysis, we started in vitro expression of “potentially” interesting salivary proteins. We initially focused our attention on two small putative proteins sharing similarity with anticoagulants from distantly related species: (a) gSG6 shows 24% identity and 65% similarity to AcAP6, an anti-coagulant from the haematophagous nematode Ancylostoma caninum that contains a TIL domain (Trypsin-Inhibitor-Like cystein-rich domain), typical of serine protease inhibitors; (b) gSG7 shares 19% identity and 45% similarity with a secreted phospholipase A2 from the venom of the cobra Naja naja atra that also possesses anticoagulant activity. The Pichia pastoris expression system was chosen because it combines advantages of prokaryotic and eukaryotic systems. The cDNAs encoding gSG6 and gSG7 were placed under control of the methanol-inducible alcohol oxidase promoter (AOX1) and a c-myc epitope and a six-histidine tag were included to facilitate detection and purification. Small scale expression trials in shake-flask were unsuccessful and, in the attempt to enhance the production, high-cell density cultures in a benchtop fermentor were performed. In this way it was possible to obtain and purify small amounts of the two proteins and use them for a few functional assays. Classical Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) tests indicated that recombinant gSG6 and gSG7 do not seem to affect either the extrinsic or the intrinsic pathway of the coagulation cascade. Since salivary secretions of haematophagous arthropods are known to contain also immuno-modulators we are presently testing the ability of these recombinant proteins to affect differentiation and maturation of dendritic cells

The pattern recognition receptor PTX3 is recruited at the synapse between dying and dendritic cells, and edits the cross-presentation of self, viral, and tumor antigens

Pentraxins are soluble pattern recognition receptors with a dual role: protection against extracellular microbes and autoimmunity. The mechanisms by which they accomplish these tasks are not yet fully understood. Here we show that the prototypic long pentraxin PTX3 is specifically recruited at both sides of the phagocytic synapse between dendritic cells (DCs) and dying cells and remains stably bound to the apoptotic membranes (estimated half-time > 36 hours). Apoptotic cells per se influence the production of PTX3 by maturing DCs. When both microbial stimuli and dying cells are present, PTX3 behaves as a flexible adaptor of DC function, regulating the maturation program and the secretion of soluble factors. Moreover a key event associated with autoimmunity (ie, the cross-presentation of epitopes expressed by apoptotic cells to T cells) abates in the presence of PTX3, as evaluated using self, viral, and tumor-associated model antigens (vinculin, NS3, and MelanA/MART1). In contrast, PTX3 did not influence the presentation of exogenous soluble antigens, an event required for immunity against extracellular pathogens. These data suggest that PTX3 acts as a third-party agent between microbial stimuli and dying cells, contributing to limit tissue damage under inflammatory conditions and the activation of autoreactive T cells. © 2006 by The American Society of Hematology