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Luigi Mario CASTELLO: Research interests/expertises

Development and validation of a specific E.L.I.S.A. to measure the gamma-carboxylated fraction of the Gas6 protein

In the recent years Gas6 protein (growth arrest specific protein 6) has been studied to understand its mechanism of action in physiological and pathological conditions. It has been shown to be involved in many pathological processes including inflammation, cancer, sepsis and stroke. Different ELISA assays, including commercial, are now available for the determination of the total protein in biological fluids, but a specific method for the determination of the active fraction, gamma-carboxylated in the presence of vitamin K, is not yet available. The purpose of this project is to develop a sandwich ELISA to measure human gamma-carboxylated Gas6 protein in plasma and cell culture supernatants.

 

Study of aggregation and Gas signaling in platelets of patients treated with warfarin

Different studies have shown, in animal models, the involvement of the vitamin K dependent protein Gas6 in thrombotic processes. However its role in human platelet aggregation is not yet clarified. For this reason we propose to evaluate platelet aggregation and signaling activated by Gas6 in platelets of patients with acute thrombotic disease. We are studying now platlets from thrombi collected directly from the coronary arteries during primary angioplasty procedures in patients with acute myocardial infarction. The platlets are studied with immunohistochemistry techniques and with western-blot. The phosphorylation status of proteins involved in pathways activated by Gas6 binding to its receptors (Tyro3, Axl and Mer) is evaluated on the same samples. Other experiments are involving patients taking warfarin, a drug that blocks gamma-carboxylation of vitamin K-dependent proteins. These experiments are conducted in vitro. Platlets are isolated and aggregation is induced by stimulation with different agonists and evaluated in the presence or absence of exogenous gamma-carboxylated Gas6.

 

Potential role of Gas6/Mer and TPO/MPL in sepsis and septic shock

Assuming that Gas6/Mer system is a physiological anti-inflammatory mechanism and that TPO / MpL system, on the contrary, has pro-inflammatory effects, our study intends to evaluate four different levels of scientific evidence of their involvement in severe sepsis and septic shock, through a process of molecular, cytological, histo-pathological and clinical analysis in animal models. The same systems will be also evaluated, traslationally, in humans. The early activation of the innate immune system, such as the TLR4 by endotoxins, determines the activation of an intracellular signal that ultimately involves the nuclear binding of a transcription factor (NFkB) and the initiation of transcription of genes for several inflammatory cytokines. Activation of inhibitory systems of this signal, as the Gas6/Mer, or the inhibition of other pro-inflammatory systems, such as TPO-Mpl, is expected to generate a reduction in the activation of NFkB with relative decrease in the expression of pro-inflammatory cytokines, which would represent an important first demonstration of the effectiveness, at least from the molecular point of view, of the modulation of inflammation by these systems. Inflammatory cytokines as IL-1, IL-6, TNF and others, are released by APC upon the recognition of an antigen on the surface of the cell and are known to be deeply involved in sustaining inflammation by activating other cell types (B and T lymphocytes). It is known that these cytokines travel in blood and are able to activate endothelial cells. The cytokine storm that has been described in the genesis of sepsis, and in the development of the more severe pictures of septic shock, is reputed to be involved in excess of activation of circulating macrophages, neutrophils and platelets. Those cells, ultimately, are probably principal actors in determining the organ failure; for these reasons, decreasing the expression of inflammatory cytokines and reducing the activation of innate immunity cells in vivo, can represent a truly measurable anti-inflammatory effect. The animal model of severe sepsis/septic shock allows us to determine whether the molecular and cellular anti-inflammatory effects of stimulating Gas6/Mer and inhibiting TPO/MPL have clinically measurable effects. The measurement of some clinical parameters, including survival, and assessment of tissue damage in target organs of sepsis (lung, heart, spleen, kidney, CNS) will allow us to prove whether the modulation of these systems has clinical efficacy in mice and may represent a possible therapeutic target for the treatment of this disease in humans.

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