IL-15 and a Two-Step Maturation Process Improve Bone Marrow-Derived Dendritic Cell Cancer Vaccine

In the last 20 years, dendritic cells (DCs) have been largely used as a platform for therapeutic vaccination in cancer patients. However, despite its proven safety and ability to induce cancer specific immune responses, the clinical benefits of DC-based immunotherapy are currently very limited. Thus, novel approaches are still needed to boost its efficacy. Our group recently showed that squaric acid treatment of antigens is an important adjuvant that can increase vaccine-induced downstream immune responses and therapeutic outcomes. Here we further improved this dendritic cell vaccine formulation by developing a new method for differentiating and maturing DCs from their bone marrow precursors. Our data demonstrate that bone marrow-derived DCs differentiated with GM-CSF and IL-15 and matured with a maturation cocktail in two steps present a more mature and immunogenic phenotype, compared to standard DC preparations. Further suppression of the prostaglandin E2 pathway achieved even more immunogenic DC phenotypes. This vaccine was more potent at delaying tumor growth, improved animal survival and induced a more immunogenic and Th1-skewed T cell response in an ovarian cancer mouse model. These promising results support future efforts for the clinical translation of this approach

An Integrated Approach to Unravelling Malaria Cell Signalling Pathways

In the current thesis we analyse protein phosphorylation pathways in P. falciparum, the protozoa responsible for the most virulent form of malaria in order to both understand the role and scope of this protein modification in the parasite, and to explore its feasibility as a new drug target. With the aim to map phosphorylation pathways controlled by P. falciparum Casein Kinase 2 (PfCK2), we developed a new chemical-biological approach based on γ-modified ATP analogues bearing reporting groups on the transferred phosphate in order to selectively tag CK2 substrates. Despite being able to efficiently synthesise a small set of analogues, the data presented here shows that the P-N linkage bond between the nucleotide and the tag is stable during the assay conditions but not during the product analysis due to its acidic liability (e.g. with HPLC, MALDI); suggesting that a different type of linkage should be chosen in the future. Detailed characterisation studies of the parasite PfCK2 presented here showed a number of important features differing from human CK2. Docking analyses with a CK2 inhibitor showed that the PfCK2 ATP binding pocket is smaller than human CK2 due to the presence of Val116 and Leu45 which in the human kinase are replaced by more bulky isoleucine residues: Ile120 and Ile49. The difference between the human and parasite CK2 orthologues extends further to mechanisms of activation and regulation. Shown here is the autophosphorylation of PfCK2 that, unlike the human orthologue, occurs within subdomain I at Thr63. This autophosphorylation is essential for full catalytic activity. In addition we also showed that Thr63 phosphorylation regulates the interaction between the calalytic α-subunit and the regulatory β2-subunit. Here, we also presented evidence for tyrosine phosphorylated proteins in parasite infected red blood cells. PfCK2 can act as a dual specificity kinase phosphorylating P. falciparum Minichromosome Maintenance protein 2 (PfMCM2) on Tyr16 in vitro. It is therefore possible that PfCK2 may contribute to tyrosine phosphorylation within the parasite. Finally, we also reported a study regarding MCM2-Ser13 phosphorylation which successfully identified PfCK1 as the kinase responsible for this event

Evoluzione geomorfologica quaternaria della conca intermontana di Carsoli (Aq)

In the present work the results of a geological and geomorphological study are presented. The study has been carried out in the Carsoli intermontane basin (AQ), a wide depression of tectonic origin, located between Abruzzo and Lazio regions, at the boundary of two different palaeogeographic domains, separated by the “Olevano-Antrodoco” tectonic line. The Carsoli basin is filled with a continental sedimentary succession of considerable thickness and extent, which has been classified into several lithostratigraphic units, mainly on the basis of their lithological, morphological and geochronological characteristics. The oldest continental sediments outcropping within the study area are of lacustrine origin (Bosco di Oricola silts, clays and sands). These sediments, whose deposition has probably occurred in the Lower – Middle Pleistocene, are characterized by a maximum thickness of about 200 m. They are widespread in the north-western and central parts of the basin (Bosco di Oricola) and are locally present on the eastern edge of the basin. The lacustrine deposits are cut across by an ancient sub-horizontal erosion surface, and at present, only some remnants remain. The lacustrine sediments are covered, on the north-eastern edge of the basin, by coarse fluvial sediments suspended on the present plain, originally deposited by the paleo-Turano River (Madonna delle Grazie gravels). In the central-western and southern part of the study area, a significant sequence of local volcanic deposits, dated around 530 – 540 kyears BP, outcrops. The volcanic sequence abruptly begins from an articulated erosive surface shaped in lacustrine deposits and Meso-Cenozoic carbonatic bedrock. Three main pyroclastic units are distinguished in the following members: 1) Oricola scalo opening–vent breccias, 2) Oricola Scalo grey tuffs, 3) S. Giovanni red tuffs. Only the last two units widely outcrops in the study area. The Oricola Scalo opening-vent breccias are relative to the early opening phase of the conduit. They consist of a massive structure connected to the depositional mechanisms of airfall and/or debris flow, separated by tuff layers related to base surge phenomena. The Oricola Scalo grey tuffs unit mainly consists of grey ash-lapilli tuffs with surge cross-laminations, and grey lapilli tuffs with a massive structure of pyroclastic flow. The overlying S. Giovanni red tuffs are characterized, instead, by a thickness and distribution greater than that of underlying grey tuffs. The unit is mainly composed of red lapilli tuffs, with dune structures or a parallel lamination of surge and airfall. After the lacustrine sedimentation and the volcanic episodes, the examined area underwent intense fluvial dynamics. This led to the sedimentation, in the central-southern and eastern part of the basin, of a sequence of alluvial deposits belonging to different depositional events. The morpho-litho-stratigraphic analysis of these fluvial deposits, in addition with radiometric ages, allowed for their classification into four units (Prati gravels, sands and silts, Fioio Stream conglomerates, Immagine gravels, sands and silts, Turano River gravels, sands and silts) progressively embedded into each other and ranging in age between the late Middle Pleistocene and the Present. Entrenched in the late Middle Pleistocene alluvial unit, in the southern sector of the basin (Fonte Bosco), a few hundred meters long and 1.5 meters thick calcareous tufa layer, dated by U-series method at 46,000 ± 6,000 years BP outcrops. With regard to the most recent depositional phases, great relevance has to be ascribed to the stratigraphic study carried out on the Fosso Luisa fan (Camerata Nuova) and the radiometric dating of colluvial horizon, there present. This dating, providing a calibrated 14C age of 3,550-3,400 years BP, has allowed for a temporal definition of some sedimentary and erosive events that marked the upper part of the fan during the final part of Holocene

Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number

Centrioles are microtubule-based organelles found in most eukaryotic cells and that are critical for the formation of cilia and flagella, as well as of centrosomes in animal cells. The number of centrioles must be strictly regulated in proliferating cells in order to ensure genome integrity upon cell division. Despite their importance, however, the mechanisms governing centriole assembly and number control remain incompletely understood, owing in part to a paucity of available small-molecule compounds for dissection and alteration of the underlying processes. Here we have developed a chemical genetic approach to identify small-molecule compounds capable of modulating centriole numbers in human cells. High-throughput screening of approximate to 2600 natural compounds identified 14 candidate molecules that either diminish (ten compounds) or augment (four compounds) the number of centrioles per cell. We investigated the mechanisms of action of four of these compounds and discovered that two of them potentially reduce centriole number through effects on NF-B signalling. Moreover, we established that one further compound blocks cell cycle progression and probably indirectly causes an augmentation of centriole number. The last compound analysed induces, in addition to excess centrioles, exceptionally long primary cilia-like structures. Overall, our analysis demonstrates that natural products constitute a rich source of tool compounds useful for unravelling and manipulating the mechanisms governing centriole assembly and number control

An integrated approach to unravelling malaria cell signalling pathways

In the current thesis we analyse protein phosphorylation pathways in P. falciparum, the protozoa responsible for the most virulent form of malaria in order to both understand the role and scope of this protein modification in the parasite, and to explore its feasibility as a new drug target. With the aim to map phosphorylation pathways controlled by P. falciparum Casein Kinase 2 (PfCK2), we developed a new chemical-biological approach based on γ-modified ATP analogues bearing reporting groups on the transferred phosphate in order to selectively tag CK2 substrates. Despite being able to efficiently synthesise a small set of analogues, the data presented here shows that the P-N linkage bond between the nucleotide and the tag is stable during the assay conditions but not during the product analysis due to its acidic liability (e.g. with HPLC, MALDI); suggesting that a different type of linkage should be chosen in the future. Detailed characterisation studies of the parasite PfCK2 presented here showed a number of important features differing from human CK2. Docking analyses with a CK2 inhibitor showed that the PfCK2 ATP binding pocket is smaller than human CK2 due to the presence of Val116 and Leu45 which in the human kinase are replaced by more bulky isoleucine residues: Ile120 and Ile49. The difference between the human and parasite CK2 orthologues extends further to mechanisms of activation and regulation. Shown here is the autophosphorylation of PfCK2 that, unlike the human orthologue, occurs within subdomain I at Thr63. This autophosphorylation is essential for full catalytic activity. In addition we also showed that Thr63 phosphorylation regulates the interaction between the calalytic α-subunit and the regulatory β2-subunit. Here, we also presented evidence for tyrosine phosphorylated proteins in parasite infected red blood cells. PfCK2 can act as a dual specificity kinase phosphorylating P. falciparum Minichromosome Maintenance protein 2 (PfMCM2) on Tyr16 in vitro. It is therefore possible that PfCK2 may contribute to tyrosine phosphorylation within the parasite. Finally, we also reported a study regarding MCM2-Ser13 phosphorylation which successfully identified PfCK1 as the kinase responsible for this event.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Polymer Nanoparticle-Mediated Delivery of Oxidized Tumor Lysate-Based Cancer Vaccines

Cancer vaccination is a powerful strategy to combat cancer. A very attractive approach to prime the immune system against cancer cells involves the use of tumor lysate as antigen source. The immunogenicity of tumor lysate can be further enhanced by treatment with hypochlorous acid. This study explores poly(lactic-co-glycolic acid) (PLGA) nanoparticles to enhance the delivery of oxidized tumor lysate to dendritic cells. Using human donor-derived dendritic cells, it is found that the use of PLGA nanoparticles enhances antigen uptake and dendritic cell maturation, as compared to the use of the free tumor lysate. The ability of the activated dendritic cells to stimulate autologous peripheral blood mononuclear cells (PBMCs) is assessed in vitro by coculturing PBMCs with A375 melanoma cells. Live cell imaging analysis of this experiment highlights the potential of nanoparticle-mediated dendritic-cell-based vaccination approaches. Finally, the efficacy of the PLGA nanoparticle formulation is evaluated in vivo in a therapeutic vaccination study using B16F10 tumor-bearing C57BL/6J mice. Animals that are challenged with the polymer nanoparticle-based oxidized tumor lysate formulation survive for up to 50 days, in contrast to a maximum of 41 days for the group that receives the corresponding free oxidized tumor lysate-based vaccine.L

Predicting combinations of immunomodulators to enhance dendritic cell-based vaccination based on a hybrid experimental and computational platform

Dendritic cell (DC)-based vaccines have been largely used in the adjuvant setting for the treatment of cancer, however, despite their proven safety, clinical outcomes still remain modest. In order to improve their efficacy, DC-based vaccines are often combined with one or multiple immunomodulatory agents. However, the selection of the most promising combinations is hampered by the plethora of agents available and the unknown interplay between these different agents. To address this point, we developed a hybrid experimental and computational platform to predict the effects and immunogenicity of dual combinations of stimuli once combined with DC vaccination, based on the experimental data of a variety of assays to monitor different aspects of the immune response after a single stimulus. To assess the stimuli behavior when used as single agents, we first developed an in vitro co-culture system of T cell priming using monocyte-derived DCs loaded with whole tumor lysate to prime autologous peripheral blood mononuclear cells in the presence of the chosen stimuli, as single adjuvants, and characterized the elicited response assessing 18 different phenotypic and functional traits important for an efficient anti-cancer response. We then developed and applied a prediction algorithm, generating a ranking for all possible dual combinations of the different single stimuli considered here. The ranking generated by the prediction tool was then validated with experimental data showing a strong correlation with the predicted scores, confirming that the top ranked conditions globally significantly outperformed the worst conditions. Thus, the method developed here constitutes an innovative tool for the selection of the best immunomodulatory agents to implement in future DC-based vaccines.status: publishe

U/Th dating of a Cladocora caespitosa from Capo S. Marco marine Quaternary deposits (Sardinia, Italy)

""A whole specimen, not reworked and well preserved of Cladocora caespitosa has been found within the marine Quaternary. deposits, outcropping along the eastern coast of the Capo San Marco Promontory. The U\\\/Th dating of this sample has provided a minimum. age of 70 ± 4 ka B.P. This dating allows to state that these marine deposits, containing the coral, are not Holocene in age."