Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.

Effects of different nutritional strategies on intestinal inflammation in pigs

Intestinal health is essential for the health of the body since the gastro-intestinal mucosa is the main site of interaction with the external environment, as well as the major area colonized by the microbiota. Intestinal health relies on proper barrier function, epithelial integrity and related mechanisms of protection (mucous layer, tight junctions, immune and inflammatory system). In pigs, during the weaning transition, intestinal inflammation and barrier integrity play a crucial role in regulating intestinal health and, consequently, pig’s health, growth and productivity. The aim of the project was to assess the impact of different nutritional strategies on the intestinal health of weaning piglets with reference to the inflammatory status and epithelial integrity. Therefore, in vivo trials were conducted to test the in-feed supplementation with zinc, tributyrin, or organic acids and nature-identical compounds (NIC) to weaning piglets. All the dietary interventions positively impacted the intestinal inflammatory status and, as a consequence, improved epithelial integrity by modulating tight junctions proteins (zinc or tributyrin) or by enhancing barrier properties measured with Ussing chambers (organic acids and NIC). These findings highlight that intestinal inflammation and barrier function are strictly linked, and that the control of inflammation is essential for adequate barrier function. In addition, in zinc trial and organic acids and NIC trial, better intestinal health could successfully result in better growth performance, as aimed for pig production improvement. To conclude, this work shows that dietary supplementation with bio-active substances such as zinc, tributyrin or organic acids and NIC may improve intestinal health of weaning piglets modulating intestinal inflammatory stress and barrier integrity and allowing better piglet’s health, growth and productivity.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.

Patterns of long-term settlement and land exploitation in the region of the Nile First Cataract (Egypt)

The First Cataract region (Egypt) has always played a crucial role as a border area and a crossroads for cultures and people living in adjacent landscapes. The region has its central point in the modern city of Aswan, but it extends up to the Kom Ombo Plain in the north and reaches the Bab el-Kalabsha in the south. Its eastern and western limits cannot be defined with the same precision, given that they are located in deserts. This research focused on the landscape analysis of the region intended as a complex entanglement of archaeological evidence in a geographical and natural environment whose changes impacted and, simultaneously, were influenced by human activities. Settlement patterns and land use can give interesting information on how these relationships worked from a diachronic perspective and how they shaped the region’s characteristics. To understand the links between the human presence and its evidence and the landscape of the First Cataract region, the integration of various datasets was needed, from historical and archaeological ones to the remote sensing observation of large areas. An area corresponding to ca. 18.000 km2 has been selected for this research. The chronological framework has been chosen to cover a considerable period, from the beginning of the 5th millennium BCE to the 7th century AD. Multi-temporality and multifunctionality appear as two essential aspects when the archaeological evidence of the First Cataract region is considered in its geographical and topographical setting as a general context for settlement patterns and resource exploitation analyses. A combination of remote sensing data and topographical materials has been integrated with archaeological evidence to obtain information about resource exploitation strategies and settlement adaptation from a diachronic perspective.